Nucleic acids comprising regions of the rat PEG-3 promoter that display elevated expression in human cancer cells and uses thereof

ABSTRACT

This invention provides an isolated nucleic acid comprising a PEG-3 promoter comprising the nucleotide sequence of −270 to +194 of FIG. 2. The invention also provides a method for identifying an agent that modulates PEG-3 promoter activity using a cell which comprises a PEG-3 promoter operatively linked to a reporter gene, wherein reduced reporter gene expression in the presence of the agent is indicative of an agent that inhibits PEG-3 promoter activity and wherein increased reporter gene expression in the presence of the agent is indicative of an agent that enhances PEG-3 promoter activity. The invention provides a method for treating cancer in a subject which comprises administering a nucleic acid comprising a PEG-3 promoter operatively linked to a gene-of-interest, wherein the gene-of-interest is selectively expressed in cancerous cells in the subject and such expression results in growth suppression or death of the cancerous cells.

[0001] The invention disclosed herein was made with Government supportunder National Cancer Institute Grant Nos. CA35675 and CA74468 from theU.S. Department of Health and Human Services. Accordingly, the U.S.Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0002] Throughout this application, various publications are referencedby author and date within the text. Full citations for thesepublications may be found listed alphabetically at the end of thespecification immediately preceding the claims. All patents, patentapplications and publications cited herein, whether supra or infra, arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of theinvention described and claimed herein.

SUMMARY OF THE INVENTION

[0003] This invention provides for an isolated nucleic acid comprising aPEG-3 promoter comprising the nucleotide sequence beginning with theguanosine (G) at position −270 and ending with the cytosine (C) atposition +194 of SEQ ID NO: 1. The invention also provides for a methodfor identifying an agent which modulates PEG-3 promoter activity in acell which comprises: (a) contacting the cell with the agent wherein thecell comprises a nucleic acid comprising a PEG-3 promoter operativelylinked to a reporter gene; (b) measuring the level of reporter geneexpression in the cell; and (c) comparing the expression level measuredin step (b) with the reporter gene expression level measured in anidentical cell in the absence of the agent, wherein a lower expressionlevel measured in the presence of the agent is indicative of an agentthat inhibits PEG-3 promoter activity and wherein a higher expressionlevel measured in the presence of the agent is indicative of an agentthat enhances PEG-3 promoter activity, thereby identifying an agentwhich modulates PEG-3 promoter activity in the cell. The inventionprovides for a method for treating cancer in a subject which comprisesadministering a nucleic acid comprising a PEG-3 promoter operativelylinked to a gene-of-interest wherein the gene of interest is selectivelyexpressed in cancerous cells in the subject and such expressionregulates expression of PEG-3 resulting in growth suppression or deathof the cancerous cells, thereby treating cancer in the subject.

BRIEF DESCRIPTION OF THE FIGURES

[0004] FIGS. 1A-1C: Anchorage independent growth and PEG-3 mRNA andprotein expression in normal, adenovirus-transformed and somatic cellhybrid rodent cells. (FIG. 1A) Anchorage-independent growth assays weredetermined by plating 5×10³ or 1×10⁴ cells in 0.4% agar containingmedium on top of a 0.8% agar medium containing base layer. After twoweeks growth, colonies ≧0.1 mm were enumerated using an invertedmicroscope. The results are the average of 3 independent experimentsusing triplicate samples per experiment±SD. (FIG. 1B) PEG3 mRNA levelswere determined by electrophoresing 15 μg of total cellular RNA in a1.2% agarose gel. RNA was transferred to nylon membranes and hybridizedwith a ³²P-labeled PEG-3 cDNA probe, the blot was stripped and thenrehybridized with a ³²P-labeled GAPDH probe. (FIG. 1C) PEG-3 and actinprotein levels were determined by Western blotting. Ten μg of proteinfrom each cell type was loaded onto a 10 denatured polyacrylamide geland electrophoreised for 3 hr followed by transfer to a nitrocellulosemembrane. PEG-3 protein was detected using Anti-PEG-3 antibody and actinprotein was detected by Ant-Actin antibody. Lane designation: 1 E11; 2E11-NMT; 3 E11-Ha-ras R12; 4 E11-NMT×CREF R1; 5 E11-NMT×CREF R2; 6E11-NMT×CREF F1; 7 E11-NMT×CREF F2; and 8 CREF.

[0005]FIG. 2: Sequence of the 2.0-kb PEG-3 promoter. (SEQ ID NO:1) Thisfragment was identified by 5′ DNA walking as described in Materials andMethods. The location of PEA3 and AP1 elements and the TATA boxes areindicated.

[0006]FIG. 3: Determination of the transcription start site of the PEG-3promoter. A primer complementary to the 5′ UTR region of PEG-3 mRNA (seeMaterials and Methods hereinbelow) was annealed with 4 μg of Poly A⁺RNAs from E11-NMT or E11 cells and used as a template for the primerextension assay. The conditions used for reverse transcription were asdescribed in Materials and Methods. A DNA sequencing reaction, using thesame primer and PEG-3 promoter as the template, was electrophoresed inparallel in the same gel with the primer extension reaction.

[0007]FIG. 4: Full-length PEG-3 promoter-luciferase activity in normal,adenovirus transformed and somatic cell hybrid rodent cells. Differentcell types were co-transfected with 5 μg of the FL PEG-Prom and 1 μg ofa pSV-β-galactosidase plasmid and luciferase activity was determined asdescribed in Materials and Methods 48 hr later. The results arestandardized by β-galactosidase activity and represent the average of 3independent experiments±SD. Results are expressed as fold activation incomparison with activity in E11, which represents 1 fold activation.

[0008] FIGS. 5A-5B: Mapping the regions of the PEG-3 promoter necessaryfor basal and elevated PEG-Prom expression in E11 and E11-NMT cells.(FIG. 5A) Schematic representation of deletion mutants of the PEG-Prom.Mutants were constructed as described in Materials and Methods. (FIG.5B) Fold activation of the FL-PEG-Prom (lane 1) and the various PEG-Promdeletion mutants (lanes 2 to 11) in E11 and E11-NMT cells. Foldactivation compares the FL-PEG-Prom and various deletion mutants ofPEG-Prom versus the specific PEG-Prom deletion construct (deleted atposition −40) which contains the TATA box and AP1 element. This deletionconstruct is given the arbitrary value of one. Promoter-luciferaseassays were performed as described in Materials and Methods.

[0009] FIGS. 6A-6B: Mutation analysis of the PEA3 and AP1 sites and theTATA box in the PEG-Prom. (FIG. 6A) Schematic representation of thespecific mutations in the PEG-Prom analyzed for activity in E11 andE11-NMT cells. Point mutations were made using a site-specificmutagenesis as described in Materials and Methods. (FIG. 6B) Foldactivation of the various PEG-Prom mutants in E11 and E11-NMT cells.Fold activation compares the PEG-Prom mutant (deleted at position −118)and additional mutants containing point or deletion mutations effectingthe PEA3 and AP1 sites and/or the TATA box region versus the specificPEG-Prom deletion construct (deleted at position −40) which contains awild-type TATA box and AP1 element. This latter deletion construct isgiven the arbitrary value of one. Promoterluciferase assays wereperformed as described in Materials and Methods.

[0010] FIGS. 7A-7B: Analysis of nuclear protein binding to AP1 and PEA3elements by EMSA. (FIG. 7A) AP1 and (FIG. 7B) PEA3 nucleoproteincomplexes in E11 and E11-NMT cells were identified using EMSA. Nuclearextracts were prepared from the two cell types and incubated with an AP1or PEA3 probe labeled with ³²P using γ³²P-ATP and T4 DNA kinase. Thereaction mixture was electrophoreised in a 5% non-denaturedpolyacrylamide gel as described in Materials and Methods. Arrow 1indicates supershifted AP1 (FIG. 7A) or PEA3 (FIG. 7B)DNA-protein-antibody complexes and arrow 2 indicates the AP1 (FIG. 7A)or PEA3 (FIG. 7B) DNAprotein complexes in E11 and E11-NMT cells. All ofthe samples contain nuclear extracts from either E11 or E11-NMT cells.Mut-oligo sample contains a mutated AP1 (FIG. 7A) or PEA3 (FIG. 7B)oligonucleotide. WT-Oligo sample contains a wild-type AP1 (FIG. 7A) orPEA3 (FIG. 7B) oligonucleotide. Competitor refers to the presence of a10× (10-fold) or 100× (100-fold) molar excess of unlabeled competitoroligonucleotides. cJun-Ab (FIG. 7A) and PEA3-Ab (FIG. 7B) samplescontain 1 or 5 μg of the respective antibody. Actin-Ab sample contains 5μg of anti-actin antibody.

[0011]FIG. 8: Effect of ectopic expression of cJun (AP1) and PEA3, aloneand in combination, on FL-PEG-Prom activity in E11 cells. Variousamounts (50 to 500 ng) of wild-type cJun (wtcjun), mutant TAM67 cJun(mutcjun), pcDNA3.1 (control vector), PEA3 (pEA3), pRC/RSV (controlvector), a combination of PEA3 and wild-type cJun (pEA3+wtcjun) or acombination of control vectors (pRC/RSV+pCDNA3.1) were transfected with5 μg of pGL3/PEG-Prom and 1 μg of pSV-β-galactosidase vector into E11cells. The results represent average fold activation in comparison withvector transfected E11 cells of 2 independent experiments withtriplicate samples per experiment±SD.

DETAILED DESCRIPTION OF THE INVENTION

[0012] The following several of the abbreviations used herein:progression elevated gene-3 (PEG-3); rat embryonic cells (RE cells);PEG-promoter (PEG-Prom); kilobases (kb). Throughout this application,references to specific nucleotides are to nucleotides present on thecoding strand of the nucleic acid. The following standard abbreviationsare used throughout the specification to indicate specific nucleotides:

[0013] C=cytosine A=adenosine

[0014] T=thymidine G=guanosine

[0015] This invention provides for an isolated nucleic acid comprising aPEG-3 promoter comprising the nucleotide sequence beginning with theguanosine (G) at position −270 and ending with the cytosine (C) atposition +194 of SEQ ID NO: 1.

[0016] The invention also provides for an isolated nucleic acidcomprising a fragment of the nucleotide sequence of claim 1 which is atleast 15 nucleotides in length.

[0017] In one embodiment, the nucleic acid fragment comprises

[0018] (i) a PEA3 protein binding sequence consisting of the nucleotidesequence beginning with the thymidine (T) at position −105 and endingwith the thymidine (T) at position −100 of SEQ ID NO: 1,

[0019] (ii) a TATA sequence consisting of the nucleotide sequencebeginning with the thymidine (T) at position −29 and ending with theadenosine (A) at position −24 of SEQ ID NO: 1, or

[0020] (iii) an AP1 protein binding sequence consisting of thenucleotide sequence beginning with the thymidine (T) at position +6 andending with the adenosine (A) at position +12 of the nucleotide sequenceshown in SEQ ID NO: 1.

[0021] In another embodiment, the nucleic acid comprises at least two ofthe nucleotide sequences (i) to (iii) listed above.

[0022] In another embodiment, the nucleic acid comprises the threenucleotide sequences (i) to (iii) listed above.

[0023] In another embodiment, the fragment has promoter activity.

[0024] In another embodiment, the fragment is operably linked to a geneof interest. In another embodiment, the gene of interest is a reportergene.

[0025] In another embodiment, the reporter gene encodesbeta-galactosidase, luciferase, chloramphenicol transferase or alkalinephosphatase.

[0026] In another embodiment, the gene of interest is a tumor suppressorgene, a gene whose expression causes apoptosis of a cell, or a cytotoxicgene.

[0027] The invention provides for a vector comprising at least one ofthe nucleic acids described herein. The invention also provides for ahost cell comprising this vector.

[0028] In another embodiment, the host cell is a tumor cell. In anotherembodiment, the tumor cell is a melanoma cell, a neuroblastoma cell, acervical cancer cell, a breast cancer cell, a lung cancer cell, aprostate cancer cell, a colon cancer cell or a glioblastoma multiformecell.

[0029] The invention also provides for a method for identifying an agentwhich modulates PEG-3 promoter activity in a cell which comprises: (a)contacting the cell with the agent wherein the cell comprises a nucleicacid comprising a PEG-3 promoter operatively linked to a reporter gene;(b) measuring the level of reporter gene expression in the cell; and (c)comparing the expression level measured in step (b) with the reportergene expression level measured in an identical cell in the absence ofthe agent, wherein a lower expression level measured in the presence ofthe agent is indicative of an agent that inhibits PEG-3 promoteractivity and wherein a higher expression level measured in the presenceof the agent is indicative of an agent that enhances PEG-3 promoteractivity, thereby identifying an agent which modulates PEG-3 promoteractivity in the cell.

[0030] In another embodiment, the cell is a melanoma cell, aneuroblastoma cell, a cervical cancer cell, a breast cancer cell, a lungcancer cell a prostate cancer cell, a colon cancer cell or aglioblastoma multiforme cell.

[0031] In another embodiment, the agent comprises a molecule having amolecular weight of about 7 kilodaltons or less.

[0032] In another embodiment, the agent is an antisense nucleic acidcomprising a nucleotide sequence complementary to at least a portion ofthe sequence shown in SEQ ID NO: 1 and is at least 15 nucleotides inlength.

[0033] In another embodiment, the agent is a DNA molecule, acarbohydrate, a glycoprotein, a transcription factor protein or adouble-stranded RNA molecule.

[0034] In another embodiment, the agent is a synthetic nucleotidesequence, a peptidomimetic, or an organic molecule having a molecularweight from 0.1 kilodaltons to 10 kilodaltons.

[0035] In another embodiment, the reporter gene encodesbeta-galactosidase, luciferase, chloramphenicol transferase or alkalinephosphatase.

[0036] In another embodiment, expression of PEG-3 promoter activitymeasured is equal to or greater than a 2.5 to 3.5 fold increase ordecrease.

[0037] The invention provides for a method for treating cancer in asubject which comprises administering a nucleic acid comprising a PEG-3promoter operatively linked to a gene-of-interest wherein the gene ofinterest is selectively expressed in cancerous cells in the subject andsuch expression regulates expression of PEG-3 resulting in growthsuppression or death of the cancerous cells, thereby treating cancer inthe subject.

[0038] In one embodiment of this invention, the nucleic acid consistsessentially of (i) a PEA3 protein binding sequence consisting of thenucleotide sequence beginning with the thymidine (T) at position −105and ending with the thymidine (T) at position −100 of SEQ ID NO: 1, (ii)a TATA sequence consisting of the nucleotide sequence beginning with thethymidine (T) at position −29 and ending with the adenosine (A) atposition −24 of SEQ ID NO: 1, and (iii) an AP1 protein binding sequenceconsisting of the nucleotide sequence beginning with the thymidine (T)at position +6 and ending with the adenosine (A) at position +12 of thenucleotide sequence shown in SEQ ID NO: 1.

[0039] In another embodiment, the nucleic acid has a sequencecomplementary to at least a portion of SEQ ID NO: 1 of at least 25nucleotides in length.

[0040] In another embodiment, the cancer is melanoma, neuroblastoma,astrocytoma, glioblastoma multiforme, cervical cancer, breast cancer,colon cancer, prostate cancer, osteoscarcoma or chrondosarcoma.

[0041] In another embodiment, the administering is carried out viainjection, oral administration, topical administration, adenovirusinfection, liposome-mediated transfer, topical application to the cellsof the subject, or microinjection.

[0042] In another embodiment, the subject is a mammal. In anotherembodiment, the mammal is a human. In another embodiment, the gene ofinterest is an gene whose expression causes apoptosis of a cell.

[0043] In another embodiment, the gene comprises an Mda-7 gene or a p53gene. In another embodiment, the gene of interest is a tumor suppressorgene. In another embodiment, the suppressor gene is mda-7. In anotherembodiment, the gene of interest is a cytotoxic gene. In anotherembodiment, expression of the cytotoxic gene causes cell death.

[0044] In another embodiment, the cytotoxic gene is selected from thegroup consisting of HSV-TK, p21, p27, and p10.

[0045] The practice of the present invention will employ, unlessotherwise indicated, conventional techniques of molecular biology,microbiology, virology, recombinant DNA technology, and immunology,which are within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Sambrook, Fritsch & Maniatis,Molecular Cloning: A Laboratory Manual, Second Edition (1989); DNACloning, Vols. I and II (D. N. Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed. 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed.1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., APractical Guide to Molecular Cloning (1984); the series, Methods InEnzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); andHandbook of Experimental Immunology, Vols. I-IV (D. M. Weir and C. C.Blackwell eds., 1986, Blackwell Scientific Publications).

[0046] As used in this specification and the appended claims, thesingular forms “a,” “an” and “the” include plural references unless thecontent clearly dictates otherwise.

[0047] The invention provides for a host cell comprising the recombinantexpression construct as described herein.

[0048] In another embodiment of the invention, the host cell is stablytransformed with the recombinant expression construct described herein.In another embodiment of the invention, the host cell is a tumor cell.

[0049] In another embodiment of the invention, the host cell is amelanocyte. In another embodiment of the invention, the cell is animmortalized cell.

[0050] In another embodiment of the invention, the tumor cell is amelanoma cell, a neuroblastoma cell, an astrocytoma cell, aglioblastomoa multifore cell, a cerival cancer cell, a breast cancercell, a lung cancer cell or a prostate cancer cell.

[0051] The invention provides for a method for expressing foreign DNA ina host cell comprising: introducing into the host cell a gene transfervector comprising a PEG-3 promoter nucleotide sequence operably linkedto a foreign DNA encoding a desired polypeptide or RNA, wherein saidforeign DNA is expressed.

[0052] In another embodiment of the invention, the gene transfer vectorencodes and expresses a reporter molecule.

[0053] In another embodiment of the invention, the reporter molecule isselected from the group consisting of beta-galactosidase, luciferase andchloramphenicol acetyltransferase.

[0054] In another embodiment of the invention, the “introducing” iscarried out by a means selected from the group consisting of adenovirusinfection, liposome-mediated transfer, topical application to the cell,and microinjection.

[0055] In another embodiment of the invention, the cancer is melanoma,neuroblastoma, astrocytoma, glioblastoma multiforme, cervical cancer,breast cancer, colon cancer, prostate cancer, osteoscarcoma, orchrondosarcoma.

[0056] In another embodiment of the invention, the cancer is a cancer ofthe central nervous system of the subject.

[0057] In another embodiment of the invention, the administering iscarried out via injection, oral administration, or topicaladministration.

[0058] In another embodiment of the invention, the carrier is an aqueouscarrier, a liposome, or a lipid carrier.

[0059] Definitions

[0060] As used herein “therapeutic gene” means DNA encoding an aminoacid sequence corresponding to a functional protein capable of exertinga therapeutic effect on cancer cells or having a regulatory effect onthe expression of a gene which functions in cells.

[0061] As used herein “nucleic acid molecule” includes both DNA and RNAand, unless otherwise specified, includes both double-stranded andsingle-stranded nucleic acids. Also included are hybrids such as DNA-RNAhybrids. Reference to a nucleic acid sequence can also include modifiedbases as long as the modification does not significantly interfereeither with binding of a ligand such as a protein by the nucleic acid orWatson-Crick base pairing.

[0062] As used herein “enhancer element” is a nucleotide sequence thatincreases the rate of transcription of the therapeutic genes or genes ofinterest but does not have promoter activity. An enhancer can be movedupstream, downstream, and to the other side of a promoter withoutsignificant loss of activity.

[0063] Two DNA or polypeptide sequences are “substantially homologous”when at least about 80% (preferably at least about 90%, and mostpreferably at least about 95%-99%) of the nucleotides or amino acidsmatch over a defined length of the molecule. As used herein,“substantially homologous” also refers to sequences showing identity(100% identical sequence) to the specified DNA or polypeptide sequence.DNA sequences that are substantially homologous can be identified in aSouthern hybridization, experiment under, for example, stringentconditions, as defined for that particular system. Defining appropriatehybridization conditions is within the skill of the art. See, e.g.,Sambrook et al., supra; DNA Cloning, vols I & II, supra; Nucleic AcidHybridization, supra.

[0064] A sequence “functionally equivalent” to a PEG-3 promoter sequenceis one which functions in the same manner as the PEG-3 promotersequence. Thus, a promoter sequence “functionally equivalent” to thePEG-3 promoter described herein is one which is capable of directingtranscription of a downstream coding sequence in substantially similartime-frames of expression and in substantially similar amounts and withsubstantially similar tissue specificity as the PEG-3 promoter sequence.

[0065] A DNA “coding sequence” or a “nucleotide sequence encoding” aparticular protein, is a DNA sequence which is transcribed andtranslated into a polypeptide in vivo or in vitro when placed under thecontrol of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′-(amino)terminus and a translation stop codon at the 3′-(carboxy) terminus. Acoding sequence can include, but is not limited to, procaryoticsequences, cDNA from eucaryotic mRNA, genomic DNA sequences fromeucaryotic (e.g., mammalian) sources, viral RNA or DNA, and evensynthetic nucleotide sequences. A transcription termination sequencewill usually be located 3′ to the coding sequence.

[0066] DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, enhancers, and the like, untranslatedregions, including 5′-UTRs (untranslated regions) and 3′-UTRs, whichcollectively provide for the transcription and translation of a codingsequence in a host cell.

[0067] “Operably linked” refers to an arrangement of nucleotide sequenceelements wherein the components so described are configured so as toperform their usual function. Thus, control sequences operably linked toa coding sequence are capable of effecting the expression of the codingsequence. The control sequences need not be contiguous with the codingsequence, so long as they function to direct the expression thereof.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence andthe promoter sequence can still be considered “operably linked” to thecoding sequence.

[0068] A control sequence “directs the transcription” of a codingsequence in a cell when RNA polymerase will bind the promoter sequenceand transcribe the coding sequence into mRNA, which is then translatedinto the polypeptide encoded by the coding sequence.

[0069] A cell has been “transformed” by exogenous DNA when suchexogenous DNA has been introduced inside the cell membrane. ExogenousDNA may or may not be integrated (covalently linked) into chromosomalDNA making up the genome of the cell. In procaryotes and yeasts, forexample, the exogenous DNA may be maintained on an episomal element,such as a plasmid. In eucaryotic cells, a stably transformed cell isgenerally one in which the exogenous DNA has become integrated into thechromosome so that it is inherited by daughter cells through chromosomereplication, or one which includes stably maintained extrachromosomalplasmids. This stability is demonstrated by the ability of theeucaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the exogenous DNA.

[0070] A “heterologous” region of a DNA construct is an identifiablesegment of DNA within or attached to another DNA molecule that is notfound in association with the other molecule in nature. For example, asequence encoding a protein other than a PEG-3 protein is considered aheterologous sequence when linked to a PEG-3 promoter. Another exampleof a heterologous coding sequence is a construct where the codingsequence itself is not found in nature (e.g., synthetic sequences havingcodons different from the native gene). Likewise, a chimeric sequence,comprising a heterologous gene linked to a PEG-3 promoter, will beconsidered heterologous since such chimeric constructs are not normallyfound in nature. Allelic variation or naturally occurring mutationalevents do not give rise to a heterologous region of DNA, as used herein.

[0071] Vectors

[0072] Especially preferred are virus based vectors. In the case ofeukaryotic cells, retrovirus or adenovirus based vectors are preferred.Such vectors contain all or a part of a viral genome, such as long termrepeats (“LTRs”), promoters (e.g., CMV promoters, SV40 promoter, RSVpromoter), enhancers, and so forth. When the host cell is a prokaryote,bacterial viruses, or phages, are preferred. Exemplary of such vectorsare vectors based upon, e.g., lambda phage. In any case, the vector maycomprise elements of more than one virus.

[0073] The resulting vectors are transfected or transformed into a hostcell, which may be eukaryotic or prokaryotic.

[0074] The gene transfer vector of the present invention mayadditionally comprise a gene encoding a marker or reporter molecule tomore easily trace expression of the vector.

[0075] The particular reporter molecule which can be employed in thepresent invention is not critical thereto. Examples of such reportermolecules which can be employed in the present invention are well-knownin the art and include beta-galactosidase (Fowler et al, Proc. Natl.Acad. Sci., USA, 74:1507 (1977)), luciferase (Tu et al, Biochem.,14:1970 (1975)), and chloramphenicol acetyltransferase (Gorman et al,Mol. Cell Biol., 2:1044-1051 (1982)).

[0076] The gene transfer vector may contain more than one gene encodingthe same or different foreign polypeptides or RNAs.

[0077] The gene transfer vector may be any construct which is able toreplicate within a host cell and includes plasmids, DNA viruses,retroviruses, as well as isolated nucleotide molecules.Liposome-mediated transfer of the gene transfer vector may also becarried out in the present invention.

[0078] Examples of such plasmids which can be employed in the presentinvention include pGL3-based plasmids (Promega™). An example of such DNAviruses which can be employed in the present invention are adenoviruses.

[0079] Adenoviruses have attracted increasing attention as expressionvectors, especially for human gene therapy (Berkner, Curr. Top.Microbiol. Immunol., 158:39-66 (1992)).

[0080] Examples of such adenovirus serotypes which can be employed inthe present invention are well-known in the art and include more than 40different human adenoviruses, e.g., Ad12 (subgenus A), Ad3 and Ad7(Subgenus B), Ad2 and Ads (Subgenus C), Ad8 (Subgenus D), Ad4 (SubgenusE), Ad40 (Subgenus F) (Wigand et al, In: Adenovirus DNA, Doerfler, Ed.,Martinus Nijhoff Publishing, Boston, pp. 408-441 (1986)). Ad5 ofsubgroup C is the preferred adenovirus employed in the presentinvention. This is because Ads is a human adenovirus about which a greatdeal of biochemical and genetic information is known, and it hashistorically been used for most constructions employing adenovirus as avector. Also, adenoviral vectors are commercially available, e.g., pCA3(Microbix Biosystems Inc.).

[0081] Methods for producing adenovirus vectors are well-known in theart (Berkner et al, Nucleic Acids Res., 11:6003-6020 (1983); van Dorenet al, Mol. Cell. Biol., 4:1653-1656 (1984); Ghosh-Choudhury et al,Biochem. Biophys. Res. Commun., 147:964-973 (1987); McGrory et al,Virol., 163:614-617 (1988); and Gluzman et al, In: Eurkaryotic ViralVectors, Ed. Gluzman, Y. pages 187-192, Cold Spring Harbor Laboratory(1982)).

[0082] Derivative Nucleic Acid Molecules

[0083] Derivative molecules would retain the functional property of thePEG-3 promoter, namely, the molecule having such substitutions willstill permit the tissue specific expression of the gene of interest.Modification is permitted so long as the derivative molecules retain itsincreased potency compared to PEG-3 promoter alone and its tissuespecificity.

[0084] Examples of therapeutic genes include suicide genes. These aregenes sequences the expression of which produces a protein or agent thatinhibits melanoma tumor cell growth or induces melanoma tumor celldeath. Suicide genes include genes encoding enzymes, oncogenes, tumorsuppressor genes, genes encoding toxins, genes encoding cytokines, or agene encoding oncostatin. The purpose of the therapeutic gene is toinhibit the growth of or kill skin cancer cells or produce cytokines orother cytotoxic agents which directly or indirectly inhibit the growthof or kill the cancer cell.

[0085] Suitable enzymes include thymidine kinase (TK), xanthine-guaninephosphoribosyltransferase (GPT) gene from E. coli or E. coli cytosinedeaminase (CD), or hypoxanthine phosphoribosyl transferase (HPRT).

[0086] Suitable oncogenes and tumor suppressor genes include neu, EGF,ras (including H, K, and N ras), p53, Retinoblastoma tumor suppressorgene (Rb), Wilm's Tumor Gene Product, Phosphotyrosine Phosphatase(PTPase), and nm23. Suitable toxins include Pseudomonas exotoxin A andS; diphtheria toxin (DT); E. coli LT toxins, Shiga toxin, Shiga-liketoxins (SLT-1, -2), ricin, abrin, supporin, and gelonin.

[0087] Suitable cytokines include interferons, GM-CSF interleukins,tumor necrosis factor (TNF) (Wong G, et al., Human GM-CSF: Molecularcloning of the complementary DNA and purification of the natural andrecombinant proteins. Science 1985; 228:810); WO9323034 (1993);Horisberger M. A., et al., Cloning and sequence analyses of cDNAs forinterferon-beta and virus-induced human Mx proteins reveal that theycontain putative guanine nucleotide-binding sites: functional study ofthe corresponding gene promoter. Journal of Virology, 1990 March,64(3):1171-81; Li Y P et al., Proinflammatory cytokines tumor necrosisfactor-alpha and IL-6, but not IL-1, down-regulate the osteocalcin genepromoter. Journal of Immunology, Feb. 1, 1992, 148(3):788-94; Pizarro T.T., et al. Induction of TNF alpha and TNF beta gene expression in ratcardiac transplants during allograft rejection. Transplantation, 1993August, 56(2):399-404). (Breviario F., et al., Interleukin-1-induciblegenes in endothelial cells. Cloning of a new gene related to C-reactiveprotein and serum amyloid P component. Journal of Biological Chemistry,Nov. 5, 1992, 267(31):22190-7; Espinoza-Delgado I., et al., Regulationof IL-2 receptor subunit genes in human monocytes. Differential effectsof IL-2 and IFN-gamma. Journal of Immunology, Nov. 1, 1992,149(9):2961-8; Algate P. A., et al., Regulation of the interleukin-3(IL-3) receptor by IL-3 in the fetal liver-derived FL5.12 cell line.Blood, 1994 May 1, 83(9):2459-68; Cluitmans F. H., et al., IL-4down-regulates IL-2-, IL-3-, and GM-CSF-induced cytokine gene expressionin peripheral blood monocytes. Annals of Hematology, 1994 June,68(6):293-8; Lagoo, A. S., et al., IL-2, IL-4, and IFN-gamma geneexpression versus secretion in superantigen-activated T cells. Distinctrequirement for costimulatory signals through adhesion molecules.Journal of Immunology, Feb. 15, 1994, 152(4):1641-52; Martinez O. M., etal., IL-2 and IL-5 gene expression in response to alloantigen in liverallograft recipients and in vitro. Transplantation, 1993 May,55(5):1159-66; Pang G, et al., GM-CSF, IL-1 alpha, IL-1 beta, IL-6,IL-8, IL-10, ICAM-1 and VCAM-1 gene expression and cytokine productionin human duodenal fibroblasts stimulated with lipopolysaccharide, IL-1alpha and TNF-alpha. Clinical and Experimental Immunology, 1994 June,96(3):437-43; Ulich T. R., et al., Endotoxin-induced cytokine geneexpression in vivo. III. IL-6 mRNA and serum protein expression and thein vivo hematologic effects of IL-6. Journal of Immunology, Apr. 1,1991, 146(7):2316-23; Mauviel A., et al., Leukoregulin, a T cell-derivedcytokine, induces IL-8 gene expression and secretion in human skinfibroblasts. Demonstration and secretion in human skin fibroblasts.Demonstration of enhanced NF-kappa B binding and NF-kappa B-drivenpromoter activity. Journal of Immunology, Nov. 1, 1992, 149(9):2969-76).

[0088] Growth factors include Transforming Growth Factor-.alpha.(TGF-alpha) and beta (TGF-beta), cytokine colony stimulating factors(Shimane M., et al., Molecular cloning and characterization of G-CSFinduced gene cDNA. Biochemical and Biophysical Research Communications,Feb. 28, 1994, 199(1):26-32; Kay A. B., et al., Messenger RNA expressionof the cytokine gene cluster, interleukin 3 (IL-3), IL-4, IL-5, andgranulocyte/macrophage colony-stimulating factor, in allergen-inducedlate-phase cutaneous reactions in atopic subjects. Journal ofExperimental Medicine, Mar. 1, 1991, 173(3):775-8; de Wit H, et al.,Differential regulation of M-CSF and IL-6 gene expression in monocyticcells. British Journal of Haematology, 1994 February, 86(2):259-64;Sprecher E., et al., Detection of IL-1 beta, TNF-alpha, and IL-6 genetranscription by the polymerase chain reaction in keratinocytes,Langerhans cells and peritoneal exudate cells during infection withherpes simplex virus-1. Archives of Virology, 1992, 126(1-4):253-69).

[0089] Preferred vectors for use in the methods of the present inventionare viral including adenoviruses, retroviral, vectors, adeno-associatedviral (AAV) vectors.

[0090] The viral vector selected should meet the following criteria: 1)the vector must be able to infect the tumor cells and thus viral vectorshaving an appropriate host range must be selected; 2) the transferredgene should be capable of persisting and being expressed in a cell foran extended period of time; and 3) the vector should be safe to the hostand cause minimal cell transformation. Retroviral vectors andadenoviruses offer an efficient, useful, and presently thebest-characterized means of introducing and expressing foreign genesefficiently in mammalian cells. These vectors have very broad host andcell type ranges, express genes stably and efficiently. The safety ofthese vectors has been proved by many research groups. In fact many arein clinical trials.

[0091] Other virus vectors that may be used for gene transfer into cellsfor correction of disorders include retroviruses such as Moloney murineleukemia virus (MoMuLV); papovaviruses such as JC, SV40, polyoma,adenoviruses; Epstein-Barr Virus (EBV); papilloma viruses, e.g. bovinepapilloma virus type I (BPV); vaccinia and poliovirus and other humanand animal viruses.

[0092] Adenoviruses have several properties that make them attractive ascloning vehicles (Bachettis et al.: Transfer of gene for thymidinekinase-deficient human cells by purified herpes simplex viral DNA. PNASUSA, 1977 74:1590; Berkner, K. L.: Development of adenovirus vectors forexpression of heterologous genes. Biotechniques, 1988 6:616;Ghosh-Choudhury G., et al., Human adenovirus cloning vectors based oninfectious bacterial plasmids. Gene 1986; 50:161; Hag-Ahmand Y., et al.,Development of a helper-independent human adenovirus vector and its usein the transfer of the herpes simplex virus thymidine kinase gene. JVirol 1986; 57:257; Rosenfeld M., et al., Adenovirus-mediated transferof a recombinant alpha..sub.1-antitrypsin gene to the lung epithelium invivo. Science 1991; 252:431).

[0093] For example, adenoviruses possess an intermediate sized genomethat replicates in cellular nuclei; many serotypes are clinicallyinnocuous; adenovirus genomes appear to be stable despite insertion offoreign genes; foreign genes appear to be maintained without loss orrearrangement; and adenoviruses can be used as high level transientexpression vectors with an expression period up to 4 weeks to severalmonths. Extensive biochemical and genetic studies suggest that it ispossible to substitute up to 7-7.5 kb of heterologous sequences fornative adenovirus sequences generating viable, conditional,helper-independent vectors (Kaufman R. J.; identification of thecomponent necessary for adenovirus translational control and theirutilization in cDNA expression vectors. PNAS USA, 1985 82:689).

[0094] AAV is a small human parvovirus with a single stranded DNA genomeof approximately 5 kb. This virus can be propagated as an integratedprovirus in several human cell types. AAV vectors have several advantagefor human gene therapy. For example, they are trophic for human cellsbut can also infect other mammalian cells; (2) no disease has beenassociated with AAV in humans or other animals; (3) integrated AAVgenomes appear stable in their host cells; (4) there is no evidence thatintegration of AAV alters expression of host genes or promoters orpromotes their rearrangement; (5) introduced genes can be rescued fromthe host cell by infection with a helper virus such as adenovirus.

[0095] HSV-1 vector system facilitates introduction of virtually anygene into non-mitotic cells (Geller et al. an efficient deletion mutantpackaging system for a defective herpes simplex virus vectors: Potentialapplications to human gene therapy and neuronal physiology. PNAS USA,1990 87:8950).

[0096] Another vector for mammalian gene transfer is the bovinepapilloma virus-based vector (Sarver N, et al., Bovine papilloma virusDNA: A novel eukaryotic cloning vector. Mol Cell Biol 1981; 1:486).Vaccinia and other poxvirus-based vectors provide a mammalian genetransfer system. Vaccinia virus is a large double-stranded DNA virus of120 kilodaltons (kd) genomic size (Panicali D, et al., Construction ofpoxvirus as cloning vectors: Insertion of the thymidine kinase gene fromherpes simplex virus into the DNA of infectious vaccine virus. Proc NatlAcad Sci USA 1982; 79:4927; Smith et al. infectious vaccinia virusrecombinants that express hepatitis B virus surface antigens. Nature,1983 302:490.)

[0097] Retroviruses are packages designed to insert viral genes intohost cells (Guild B, et al., Development of retrovirus vectors usefulfor expressing genes in cultured murine embryonic cells andhematopoietic cells in vivo. J Virol 1988; 62:795; Hock R. A., et al.,Retrovirus mediated transfer and expression of drug resistance genes inhuman hemopoietic progenitor cells. Nature 1986; 320:275).

[0098] The basic retrovirus consists of two identical strands of RNApackaged in a proviral protein. The core surrounded by a protective coatcalled the envelope, which is derived from the membrane of the previoushost but modified with glycoproteins contributed by the virus.

[0099] Markers and amplifiers can also be employed in the subjectexpression systems. A variety of markers are known which are useful inselecting for transformed cell lines and generally comprise a gene whoseexpression confers a selectable phenotype on transformed cells when thecells are grown in an appropriate selective medium. Such markers formammalian cell lines include, for example, the bacterialxanthine-guanine phosporibosyl transferase gene, which can be selectedfor in medium containing mycophenolic acid and xanthine (Mulligan et al.(1981) Proc. Natl. Acad. Sci. USA 78:2072-2076), and the aminoglycosidephosphotransferase gene (specifying a protein that inactivates theantibacterial action of neomycin/kanamycin derivatives), which can beselected for using medium containing neomycin derivatives such as G418which are normally toxic to mammalian cells (Colbere-Garapin et al.(1981) J. Mol. Biol. 150:1-14). Useful markers for other eucaryoticexpression systems, are well known to those of skill in the art.

[0100] Infection can be carried out in vitro or in vivo. In vitroinfection of cells is performed by adding the gene transfer vectors tothe cell culture medium. When infection is carried out in vivo, thesolution containing the gene transfer vectors may be administered by avariety of modes, depending on the tissue which is to be infected.Examples of such modes of administration include injection of genetransfer vectors into the skin, topical application onto the skin,direct application to a surface of epithelium, or instillation into anorgan (e.g., time release patch or capsule below the skin or into atumor).

[0101] Expression can be amplified by placing an amplifiable gene, suchas the mouse dihydrofolate reductase (dhfr) gene adjacent to the codingsequence. Cells can then be selected for methotrexate resistance indhfr-deficient cells. See, e.g. Urlaub et al. (1980) Proc. Natl. Acad.Sci. USA 77:4216-4220; Rungold et al. (1981) J. Mol. and Appl. Genet.1:165-175.

[0102] The above-described system can be used to direct the expressionof a wide variety of procaryotic, eucaryotic and viral proteins,including, for example, viral glycoproteins suitable for use as vaccineantigens, immunomodulators for regulation of the immune response,hormones, cytokines and growth factors, as well as proteins useful inthe production of other biopharmaceuticals.

[0103] It may also be desirable to produce mutants or analogs of theproteins of interest. Mutants or analogs may be prepared by the deletionof a portion of the sequence encoding the protein, by insertion of asequence, and/or by substitution of one or more nucleotides within thesequence. Techniques for modifying nucleotide sequences, such assite-directed mutagenesis, are well known to those skilled in the art.See, e.g., Sambrook et al., supra; DNA Cloning, Vols. I and II, supra;Nucleic Acid Hybridization, supra.

[0104] For purposes of the present invention, it is particularlydesirable to further engineer the coding sequence to effect secretion ofthe polypeptide from the host organism. This enhances clone stabilityand prevents the toxic build up of proteins in the host cell so thatexpression can proceed more efficiently. Homologous signal sequences canbe used for this purpose with proteins normally found in associationwith a signal sequence. Additionally, heterologous leader sequenceswhich provide for secretion of the protein can be added to theconstructs. Preferably, processing sites will be included such that theleader fragment can be cleaved from the protein expressed therewith.(See, e.g., U.S. Pat. No. 4,336,246 for a discussion of how suchcleavage sites can be introduced). The leader sequence fragmenttypically encodes a signal peptide comprised of hydrophobic amino acids.

[0105] In one embodiment of the invention, a heterologous gene sequence,i.e., a therapeutic gene, is inserted into the nucleic acid molecule ofthe invention. Other embodiments of the isolated nucleic acid moleculeof the invention include the addition of a single enhancer element ormultiple enhancer elements which amplify the expression of theheterologous therapeutic gene without compromising tissue specificity.

[0106] The transformation procedure used depends upon the host to betransformed. Mammalian cells can conveniently be transformed using, forexample, DEAE-dextran based procedures, calcium phosphate precipitation(Graham, F. L. and Van der Eb, A. J. (1973) Virology 52:456-467),protoplast fusion, liposome-mediated transfer, polybrene-mediatedtransfection and direct microinjection of the DNA into nuclei. Bacterialcells will generally be transformed using calcium chloride, either aloneor in combination with other divalent cations and DMSO (Sambrook,Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, SecondEdition (1989)). DNA can also be introduced into bacterial cells byelectroporation. Methods of introducing exogenous DNA into yeast hoststypically include either the transformation of spheroplasts ortransformation of intact yeast cells treated with alkali cations.

[0107] The constructs can also be used in gene therapy or nucleic acidimmunization, to direct the production of the desired gene product invivo, by administering the expression constructs directly to a subjectfor the in vivo translation thereof. See, e.g. EPA Publication No.336,523 (Dreano et al., published Oct. 11, 1989). Alternatively, genetransfer can be accomplished by transfecting the subject's cells ortissues with the expression constructs ex vivo and reintroducing thetransformed material into the host. The constructs can be directlyintroduced into the host organism, i.e., by injection (see InternationalPublication No. WO/90/11092; and Wolff et al., (1990) Science247:1465-1468). Liposome-mediated gene transfer can also be accomplishedusing known methods. See, e.g., Hazinski et al., (1991) Am. J. Respir.Cell Mol. Biol. 4:206-209; Brigham et al. (1989) Am. J. Med. Sci.298:278-281; Canonico et al. (1991) Clin. Res. 39:219A; and Nabel et al.(1990) Science 249:1285-1288. Targeting agents, such as antibodiesdirected against surface antigens expressed on specific cell types, canbe covalently conjugated to the liposomal surface so that the nucleicacid can be delivered to specific tissues and cells for localadministration.

[0108] Human Gene Therapy and Diagnostic Use of Vector

[0109] There are several protocols for human gene therapy which havebeen approved for use by the Recombinant DNA Advisory Committee (RAC)which conform to a general protocol of target cell infection andadministration of transfected cells (see for example, Blaese, R. M., etal., 1990; Anderson, W. F., 1992; Culver, K. W. et al., 1991). Inaddition, U.S. Pat. No. 5,399,346 (Anderson, W. F. et al., March 21,1995, U.S. Ser. No. 220,175) describes procedures for retroviral genetransfer. The contents of these support references are incorporated intheir entirety into the subject application. Retroviral-mediated genetransfer requires target cells which are undergoing cell division inorder to achieve stable integration hence, cells are collected from asubject often by removing blood or bone marrow. It may be necessary toselect for a particular subpopulation of the originally harvested cellsfor use in the infection protocol. Then, a retroviral vector containingthe gene(s) of interest would be mixed into the culture medium. Thevector binds to the surface of the subject's cells, enters the cells andinserts the gene of interest randomly into a chromosome. The gene ofinterest is now stably integrated and will remain in place and be passedto all of the daughter cells as the cells grow in number. The cells maybe expanded in culture for a total of 9-10 days before reinfusion(Culver et al., 1991). As the length of time the target cells are leftin culture increases, the possibility of contamination also increases,therefore a shorter protocol would be more beneficial.

[0110] This invention provides for the construction of retrovirusvectors containing the PEG-3 promoter or a functional equivalent thereoflinked to a gene of interest for use in gene therapy or for diagnosticuses. The efficiency of transduction of these vectors can be tested incell culture systems.

[0111] Uses of the Compositions of the Invention

[0112] This invention involves targeting a gene-of-interest to the acancer cell so that the protein encoded by the gene is expressed anddirectly or indirectly ameliorate the diseased state. Since the PEG-3promoter is specifically active in a cancer cell which is undergoingcancer progression, it will act as a tissue specific promoter (specificfor cancer cells).

[0113] After infecting a susceptible cell, the transgene driven by aspecific promoter in the vector expresses the protein encoded by thegene. The use of the highly specific gene vector will allow selectiveexpression of the specific genes in cancer cells.

[0114] The basic tasks in the present method of the invention areisolating the gene of interest, selecting the proper vector vehicle todeliver the gene of interest to the body, administering the vectorhaving the gene of interest into the body, and achieving appropriateexpression of the gene of interest. The present invention providespackaging the cloned genes, i.e. the genes of interest, in such a waythat they can be injected directly into the bloodstream or relevantorgans of patients who need them. The packaging will protect the foreignDNA from elimination by the immune system and direct it to appropriatetissues or cells.

[0115] In one embodiment of the invention, the gene of interest (desiredcoding sequence) is a tumor suppressor gene. The tumor suppressor genemay be p21, RB (retinoblastoma) or p53. One of skill in the art wouldknow of other tumor suppressor genes. Recent U.S. Pat. Nos. 6,025,127and 5,912,236 are hereby incorporated by reference to more explicitlydescribe the state of the art as to tumor suppressor genes.

[0116] Along with the human or animal gene of interest another gene,e.g., a selectable marker, can be inserted that will allow easyidentification of cells that have incorporated the modified retrovirus.The critical focus on the process of gene therapy is that the new genemust be expressed in target cells at an appropriate level with asatisfactory duration of expression.

[0117] The methods described below to modify vectors and administeringsuch modified vectors into the skin are merely for purposes ofillustration and are typical of those that might be used. However, otherprocedures may also be employed, as is understood in the art.

[0118] Most of the techniques used to construct vectors and the like arewidely practiced in the art, and most practitioners are familiar withthe standard resource materials which describe specific conditions andprocedures. However, for convenience, the following paragraphs may serveas a guideline.

[0119] General Methods for Vector Construction

[0120] Construction of suitable vectors containing the desiredtherapeutic gene coding and control sequences employs standard ligationand restriction techniques, which are well understood in the art (seeManiatis et al., in Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory, New York (1982)). Isolated plasmids, DNA sequences,or synthesized oligonucleotides are cleaved, tailored, and religated inthe form desired.

[0121] Site-specific DNA cleavage is performed by treating with thesuitable restriction enzyme (or enzymes) under conditions which aregenerally understood in the art, and the particulars of which arespecified by the manufacturer of these commercially availablerestriction enzymes (See, e.g. New England Biolabs Product Catalog). Ingeneral, about 1 μg of plasmid or DNA sequences is cleaved by one unitof enzyme in about 20 μl of buffer solution. Typically, an excess ofrestriction enzyme is used to insure complete digestion of the DNAsubstrate.

[0122] Incubation times of about one hour to two hours at about 37degree. C. are workable, although variations can be tolerated. Aftereach incubation, protein is removed by extraction withphenol/chloroform, and may be followed by ether extraction, and thenucleic acid recovered from aqueous fractions by precipitation withethanol. If desired, size separation of the cleaved fragments may beperformed by polyacrylamide gel or agarose gel electrophoresis usingstandard techniques. A general description of size separations is foundin Methods in Enzymology 65:499-560 (1980). Restriction cleavedfragments may be blunt ended by treating with the large fragment of E.coli DNA polymerase I (Klenow) in the presence of the fourdeoxynucleotide triphosphates (dNTPs) using incubation times of about 15to 25 min at 20.degree. C. to 25.degree. C. in 50 mM Tris (pH 7.6) 50 mMNaCl, 6 mM MgCl.sub.2, 6 mM DTT and 5-10 .mu.M dNTPs. The Klenowfragment fills in at 5′ sticky ends but chews back protruding 3′ singlestrands, even though the four dNTPs are present. If desired, selectiverepair can be performed by supplying only one of the dNTPs, or withselected dNTPs, within the limitations dictated by the nature of thesticky ends. After treatment with Klenow, the mixture is extracted withphenol/chloroform and ethanol precipitated. Treatment under appropriateconditions with S1 nuclease or Bal-31 results in hydrolysis of anysingle-stranded portion.

[0123] Ligations are performed in 10-50 μl volumes under the followingstandard conditions and temperatures using T4 DNA ligase. Ligationprotocols are standard (D. Goeddel (ed.) Gene Expression Technology:Methods in Enzymology (1991)). In vector construction employing “vectorfragments”, the vector fragment is commonly treated with bacterialalkaline phosphatase (BAP) or calf intestinal alkaline phosphatase (CIP)in order to remove the 5′ phosphate and prevent religation of thevector. Alternatively, religation can be prevented in vectors which havebeen double digested by additional restriction enzyme digestion of theunwanted fragments.

[0124] Suitable vectors include viral vector systems e.g. ADV, RV, andAAV (R. J. Kaufman “Vectors used for expression in mammalian cells” inGene Expression Technology, edited by D. V. Goeddel (1991).

[0125] Many methods for inserting functional DNA transgenes into cellsare known in the art. For example, non-vector methods include nonviralphysical transfection of DNA into cells; for example, microinjection(DePamphilis et al., BioTechnique 6:662-680 (1988)); liposomal mediatedtransfection (Felgner et al., Proc. Natl. Acad. Sci. USA, 84:7413-7417(1987), Felgner and Holm, Focus 11:21-25 (1989) and Felgner et al.,Proc. West. Pharmacol. Soc. 32: 115-121 (1989)) and other methods knownin the art.

[0126] Administration of Modified Vectors Into Subject

[0127] One way to get DNA into a target cell is to put it inside amembrane bound sac or vesicle such as a spheroplast or liposome, or bycalcium phosphate precipitation (CaPO.sub.4) (Graham F. and Van der Eb,A., Virology 52:456 1973; Schaefer-Ridder M., et al., Liposomes as genecarriers: Efficient transduction of mouse L cells by thymidine kinasegene. Science 1982; 215:166; Stavridis J. C., et al., Construction oftransferrin-coated liposomes for in vivo transport of exogenous DNA tobone marrow erythroblasts in rabbits. Exp Cell Res 1986; 164:568-572).

[0128] A vesicle can be constructed in such a way that its membrane willfuse with the outer membrane of a target cell. The vector of theinvention in vesicles can home into the cancer cells.

[0129] The spheroplasts are maintained in high ionic strength bufferuntil they can be fused through the mammalian target cell using fusogenssuch as polyethylene glycol.

[0130] Liposomes are artificial phospholipid vesicles. Vesicles range insize from 0.2 to 4.0 micrometers and can entrap 10% to 40% of an aqueousbuffer containing macromolecules. The liposomes protect the DNA fromnucleases and facilitate its introduction into target cells.Transfection can also occur through electroporation. Beforeadministration, the modified vectors are suspended in complete PBS at aselected density for injection. In addition to PBS, any osmoticallybalanced solution which is physiologically compatible with the subjectmay be used to suspend and inject the modified vectors into the host.

[0131] For injection, the cell suspension is drawn up into the syringeand administered to anesthetized recipients. Multiple injections may bemade using this procedure. The viral suspension procedure thus permitsadministration of genetically modified vectors to any predetermined sitein the skin, is relatively non-traumatic, allows multipleadministrations simultaneously in several different sites or the samesite using the same viral suspension. Multiple injections may consist ofa mixture of therapeutic genes.

[0132] Survival of the Modified Vectors So Administered

[0133] Expression of a gene is controlled at the transcription,translation or post-translation levels. Transcription initiation is anearly and critical event in gene expression. This depends on thepromoter and enhancer sequences and is influenced by specific cellularfactors that interact with these sequences. The transcriptional unit ofmany prokaryotic genes consists of the promoter and in some casesenhancer or regulator elements (Banerji et al., Cell 27:299 (1981);Corden et al., Science 209:1406 (1980); and Breathnach and Chambon, Ann.Rev. Biochem. 50:349 (1981)). For retroviruses, control elementsinvolved in the replication of the retroviral genome reside in the longterminal repeat (LTR) (Weiss et al., eds., In: The molecular biology oftumor viruses: RNA tumor viruses, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y. (1982)).

[0134] Moloney murine leukemia virus (MLV) and Rous sarcoma virus (RSV)LTRs contain promoter and enhancer sequences (Jolly et al., NucleicAcids Res. 11:1855 (1983); Capecchi et al., In: Enhancer and eukaryoticgene expression, Gulzman and Shenk, eds., pp. 101-102, Cold SpringHarbor Laboratories, Cold Spring Harbor, N.Y.).

[0135] Promoter and enhancer regions of a number of non-viral promotershave also been described (Schmidt et al., Nature 314:285 (1985); Rossiand de Crombrugghe, Proc. Natl. Acad. Sci. USA 84:5590-5594 (1987)).

[0136] In addition to using viral and non-viral promoters to drivetherapeutic gene expression, an enhancer sequence may be used toincrease the level of therapeutic gene expression. Enhancers canincrease the transcriptional activity not only of their native gene butalso of some foreign genes (Armelor, Proc. Natl. Acad. Sci. USA 70:2702(1973)).

[0137] Therapeutic gene expression may also be increased for long termstable expression after injection using cytokines to modulate promoteractivity.

[0138] The methods of the invention are exemplified by preferredembodiments in which modified vectors carrying a therapeutic gene areinjected intracerebrally into a subject.

[0139] The most effective mode of administration and dosage regimen forthe molecules of the present invention depends upon the exact locationof the cancer being treated, the severity and course of the cancer, thesubject's health and response to treatment and the judgment of thetreating physician. Accordingly, the dosages of the molecules should betitrated to the individual subject. The molecules may be delivereddirectly or indirectly via another cell, autologous cells are preferred,but heterologous cells are encompassed within the scope of theinvention.

[0140] The interrelationship of dosages for animals of various sizes andspecies and humans based on mg/m.sup.2 of surface area is described byFreireich, E. J., et al. Cancer Chemother., Rep. 50 (4):219-244 (1966).Adjustments in the dosage regimen may be made to optimize the tumor cellgrowth inhibiting and killing response, e.g., doses may be divided andadministered on a daily basis or the dose reduced proportionallydepending upon the situation (e.g., several divided dose may beadministered daily or proportionally reduced depending on the specifictherapeutic situation).

[0141] It would be clear that the dose of the molecules of the inventionrequired to achieve cures may be further reduced with scheduleoptimization.

[0142] Use of PEG-Promoter to Direct High Expression of a HeterlogousGene in Cancer Cells

[0143] One embodiment of the invention provides for methods forexpressing a gene of interest which gene is not endogenously expressedin cancer cells which comprises a) constructing a nucleic acid whichcomprises the PEG-3 promoter operatively linked to the gene-of-interest;b) introducing this nucleic acid into a cancer cell which cell expressesPEG-3, thereby causing the PEG-3 promoter to direct expression of thegene-of-interest in the cancer cell. In one embodiment, thegene-of-interest encodes a protein which is cytotoxic to the cancercell, causes apoptosis of the cancer cell, slows the growth of thecancer cell, or causes the cancer cell to stop dividing. Thegene-of-interest can be any gene whose expression would cause a desiredbiochemical or physiological effect in the cancer cell, such as thedecrease of growth or the decrease or inhibition of cancer phenotypeprogression.

[0144] One advantage of using the nucleic acid construct described abovein such a method to treat cancer in a subject, is that the nucleic acidcan be administered to both cancerous and normal cells. However, sincethe PEG-3 promoter is only active in cancerous cells, there will be noexpression of the gene-of-interest in normal cells, while there will behigh expression of the gene-of-interest in the cancerous cells. Thisnucleic acid construct thus allows one to target specifically expressionof a gene-of-interest to specifically cancerous cells.

[0145] Liposomes could be used as a delivery agent to introduce thenucleic acid construct to the cells of the subject to be treated. Ofcourse, there are many ways to deliver such a nucleic acid constructwhich would be known to one of skill in the art (e.g. microinjection;topical application; use of a chemical vehicle; direct injection intothe tumor; etc.).

[0146] This invention is illustrated in the Experimental Details sectionwhich follows. These sections are set forth to aid in an understandingof the invention but are not intended to, and should not be construedto, limit in any way the invention as set forth in the claims whichfollow thereafter.

EXPERIMENTAL DETAILS EXAMPLE 1

[0147] Defining the Regions Within the Promoter of Progression ElevatedGene-3 Responsible for Differential Expression During TransformationProgression

[0148] Cancer is a progressive disease in which a tumor cell temporallydevelops qualitatively new transformation related phenotypes or afurther elaboration of existing transformation associated properties. Arodent cell culture model system is being used to define the genes thatassociate with and control cancer progression. Subtraction hybridizationidentified a novel gene that is functionally involved in the inductionof transformation progression in mutant adenovirus type 5, H5ts125,transformed rat embryo cells, referred to as progression elevated gene-3(PEG-3). A 5′-flanking promoter region of ˜2.1 kilobases, PEG-promoter,has been isolated, cloned and characterized. The full-length and variousmutated regions of the PEG-promoter have been linked to a luciferasereporter construct and evaluated for promoter activity during cancerprogression using transient transfection assays. These experimentsdemonstrate a requirement for AP-1 and PEA-3 sites adjacent to the TATAbox region of PEG-3 in mediating enhanced expression of PEG-3 inprogressed versus un-progressed H5-ts125-transformed rat embryo cells.An involvement of AP-1 and PEA-3 in PEG-3 regulation was alsodemonstrated by protein blotting, electrophoretic mobility shift (EMSA)assays and transfection studies with PEA-3 and c-Jun expression vectors.Our findings document the importance of the AP-1 and PEA-3 transcriptionfactors in mediating elevated expression of PEG-3 in H5ts125-transformedrat embryo cells displaying an aggressive and progressed cancerphenotype.

EXAMPLE 2

[0149] Cooperation Between AP-1 and PEA-3 Sites Within the ProgressionElevated Gene-3 (PEG-3) Promoter Regulate Basal and DifferentialExpression of PEG-3 During Progression of the Oncogenic Phenotype inTransformed Rat Embryo Cells

[0150] The carcinogenic process involves a series of sequential changesin the phenotype of a cell, resulting in new properties or a furtherelaboration of transformation-associated traits by the evolving tumorcell (Fisher, 1984; Bishop, 1991; Knudson, 1993; Vogelstein and Kinzler,1993). Although extensively studied, the precise genetic mechanismsunderlying tumor cell progression during the development of most humancancers remain unknown. Experimental evidence indicates that a number ofdiverse acting genetic elements can contribute to cancer development andtransformation progression (Fisher, 1984; Bishop, 1991; Liotta et al.,1991; Knudson, 1993; Levine, 1993; Hartwell and Kastan, 1994; Kang etal., 1998a; Vogelstein and Kinzler, 1993; Su et al., 1997; 1999).Important target genes involved in these processes include, oncogenes,tumor supressor genes and genes regulating genomic stability, canceragressiveness and angiogenesis (Fisher, 1984; Bishop, 1991; Liotta etal., 1991; Knudson, 1993; Levine, 1993; Hartwell and Kastan, 1994; Kanget al., 1998a; Vogelstein and Kinzler, 1993; Su et al., 1997, 1999).Recently, several novel genetic elements have been identified thatassociate with or in specific instances directly regulate canceragressiveness, i.e. progression elevated (PEGen) and progressionsuppressed (PSGen) genes (Kang, et al., 1998a; Su et al., 1997, 1999).The precise mechanism by which these different genes orchestrate thecomplex process of cancer progression represent an important area ofinvestigation with potential for defining novel pathways and targetmolecules that could lead to new diagnostic and therapeutic approachesfor cancer.

[0151] A useful model for defining the genetic and biochemical changesmediating tumor progression is the Ad5/early passage RE cell culturesystem (Fisher, 1984; Babiss et al., 1985; Duigou et al., 1989, 1990,1991; Fisher et al, 1979a,b,c; Reddy et al., 1993; Su et al., 1994,1997; Kang et al., 1998a). Transformation of secondary rat embryo (RE)cells by Ad5 is often a sequential process resulting in the acquisitionof an further elaboration of specific phenotypes by the untransformedcell (Fisher et al., 1979 a,b,c; Babiss et al, 1985). Progression in theAd5-transformation model is characterized by the development of enhancedanchorage-independence and tumorigenic capacity (as formation in nudemice) (Fisher, 1984; Babiss et al., 1985). The progression phenotype inAd5-transformed RE cells can be induced by selection for growth in agaror tumor formation in nude mice (Fisher et al., 1979 a,b,c; Babiss etal., 1985) by transfection with oncogenes, such as Ha-ras, v-src, v-rafor the E6/E7 region of human papilloma virus type 18 (Duigou et al.,1989; Reddy et al., 1993) or by transfection with specific signaltransducing genes, such as protein kinase C (Su et al., 1994).

[0152] Progression induced spontaneously or after gene transfer, is astable cellular trait that remains undiminished in Ad5-transformed REcells even after extensive passage (>100) in monolayer culture (Fisher,1984; Babiss et al., 1985; Reddy et al., 1993). However, asingle-treatment with the demethylating agent 5-azacytidine (AZA)results in a stable reversion in transformation progression in >95% ofcellular clones (Fisher, 1984; Babiss et al., 1985; Duigou et al., 1989;Reddy et al., 1993; Su et al., 1994). The progression phenotype is alsosuppressed in somatic cell hybrids formed between normal orun-progressed transformed cells and progressed cells (Duigou et al.,1990, 1991; Reddy et al., 1993). These findings suggest that progressionmay result from the activation of specific progression-promoting(progression elevated) genes or the selective inhibition ofprogression-suppression (progression suppressed) genes, or possibly acombination of both processes (Fisher, 1984; Babiss et al., 1985; Su etal., 1997; Kang et al., 1998a). To identify potential progressioninducing genes with elevated expression in progressed versusun-progressed Ad5 transformed cells, we are using subtractionhybridization and reciprocal subtraction differential RNA display (RSDD)approaches (Jiang and Fisher, 1993; Reddy et al., 1993; Su et al., 1997;Kang et al., 1998a). The subtraction hybridization approach resulted incloning of PEG-3 which displays elevated expression in progressed cells(spontaneous, oncogene-induced or growth-factor related gene-induced)than in un-progressed cells (parental Ad5-transformed, AZA-suppressed,and suppressed somatic cell hybrids) (Su et al, 1997). These findingsdocument a direct correlation between expression of PEG-3 and theprogression phenotype in this rat embryo model system.

[0153] Nuclear run-on assays confirm a direct correlation between PEG-3expression and an increase in the rate of RNA transcription of this gene(Su et al., 1997). To elucidate the mechanism underlying thedifferential expression of PEG-3 during transformation progression the5′-flanking region of this gene which contains the promoter (PEG-Prom)has been isolated and characterized. The full-length ˜2.0 kb PEG-Promand various mutations (including deletions and point mutations) inPEGProm were constructed and analysed. The results of this inquirydemonstrate that AP1 and PEA3 transcription factors are the primarydeterminants of the elevated expression of PEG-3 in progressedAd5-transformed RE cells. This conclusion is verified by electrophoreticmobility shift assays (EMSA) and transfection studies with c-Jun andPEA3 expression vectors.

[0154] Results

[0155] Expression of PEG3 Directly Correlates With TransformationProgression

[0156] To evaluate the relationship between PEG-3 expression andtransformation progression we have used a series of rodent cell linesthat span the gamut from normal to highly progressed (Fisher et al.1987; Babiss et al., 1985; Duigou et al., 1989; Reddy, et al., 1993; Suet al., 1997, 1999). A hallmark of the progression phenotype in thisrodent model is the ability to grow with enhanced efficiency in ananchorage-independent manner and to induce tumors in nude mice with areduced tumor latency time (18-21 days as opposed to 38-44 days,respectively) (Babiss et al., 1985; Su et al, 1999). A specificH5ts125-transformed secondary Sprague-Dawley RE clone, Eli, grows inagar with low efficiency (˜2-40%) (progression negative), whereas ahighly progressed nude mouse tumor-derived E11 subclone, E11-NMT, growswith high efficiency in agar (˜30-45%) (FIG. 1A). Forced expression ofthe Ha-ras oncogene in E11 cells, E11-ras R12 as a representative clone,results in acquisition of the progression phenotype as indicated by bothanchorage-independent growth (FIG. 1A) and tumor latency time in nudemice (Reddy et al., 1993). Quantifying PEG-3 mRNA levels by Northernhybridization (FIG. 1B) and PEG-3 protein levels by Western blotting(FIG. 1C) indicates a direct correlation between PEG-3 expression,elevated in E11-NMT and E11-ras R12 and reduced in E11, and expressionof the progression phenotype (as indicated by anchorage independentgrowth).

[0157] To explore further the relationship between PEG-3 expression andprogression, the same three parameters as measured for E11, E11-NMT andE11-ras R12 cells were used to compare a series of somatic cell hybridsformed between E11-NMT and CREF cells (FIG. 1). CREF cells are immortalrat embryo cells that do not form colonies when grown in agar and aredevoid of tumorigenic potential when inoculated subcutaneously intoathymic nude mice (Fisher et al., 1982; Duigou et al., 1990). Similarly,somatic cell hybrids formed between E11-NMT and CREF cells that displaya fat morphology such as F1 and F2, also fail to form tumors in nudemice (Duigou et al., 1990), although they grow with a low efficiency inagar similar to E11 cells (FIG. 1A). In contrast, specific E11-NMT×CREFsomatic cell hybrids that display round morphology such as R1 and R2,grown with high efficiency in agar, even exceeding that of E11-NMT (FIG.1A) and they rapidly form tumors in nude mice (Duigou et al., 1990). Asobserved with E11 cells, the levels of PEG-3 mRNA and protein arereduced in F1 and F2 cells, whereas R1 and R2 display elevatedexpression of PEG-3 akin to that of E11-NMT and E11-ras R12 cells (FIGS.1B, 1C). In the case of CREF cells, PEG-3 mRNA is detected at very lowlevels by Northern blotting (FIG. 1B) and PEG-3 protein is barelydetectable by Western blotting (FIG. 1C). These results indicate adirect concordance between PEG-3 expression and the progressionphenotype in HSts125-transformed RE cells.

[0158] Isolation of the PEG-3 Promoter and Identification of theTranscription Start Site

[0159] Based on the sequence of the PEG-3 cDNA, a genomic walkingapproach from the 5′ region of the PEG-3 cDNA was used to identify a2.0-kb rat genomic fragment that represents the 5′ flanking region ofthe PEG-3 gene. The sequence of the putative FL-PEG-Prom, is shown inFIG. 2. The transcription start site of the PEG-3 gene was mapped byprimer extension with RNAs isolated from E11 and E11-NMT cells (FIG. 3).Computer analysis with GCG software of the PEG-Prom indicates thepresence of two TATA boxes located at positions −1071 and −24 upstreamof the RNA cap site, respectively. The sequence at −1071 is probablynon-functional because of its large distance from the RNA cap-site. TwoPEA3-binding sites, AGGAAA and TTTCCT, are located at positions −1644and −101. The PEA3 site at position −101 is 76 nt upstream of the TATAbox. An AP1 site is present at position +8. Additional potential DNAbinding elements are also apparent in the PEG-Prom, including Spl, acutephase reaction element, NFKB1, E2F, E2A, GRE, TRE and CREB.

[0160] AP1 and PEA3 Sites Adjacent to the TATA Box in the PEG-3 Promoterare Involved in Basal and Enhanced Promoter Activity in Progressed andUn-Progressed H5ts125transformed RE Cells

[0161] Transfection of the FL-PEG-Prom luciferase construct into thedifferent cell types demonstrated a direct relationship betweenexpression of the progression phenotype and elevated promoter activity(FIG. 4). Progressed cells displayed a 2.5- to 3.5-fold increase inluciferase activity, a value that compares well with PEG-3 Northern andWestern blotting data (FIGS. 1B and 1C). The level of luciferaseactivity in E11 cells was similar to that observed in the F1 and F2CREF×E11-NMT somatic cell hybrids. In the case of actively proliferatingCREF cells, the PEG-prom exhibited negligible activity.

[0162] To define the region(s) of the FL-PEG-Prom involved in thedifferential expression of the PEG-3 gene during progression of thetransformed phenotype in H5ts125-transformed cells, a series of PEG-Promdeletion constructs were engineered and placed in front of theluciferase gene (FIGS. 5 and 6). Deletion of the PEA3 site at position−1645 and the TATA box at position −1072 did not effect PEG promoteractivity in either E11 or E11-NMT suggesting that these regions of thepromoter do not contribute to basal or enhanced expression of thePEG-Prom in E11 or E11-NMT cells (FIG. 5). A further deletion atposition −270 minimally inhibited promoter activity in E11-NMT cells(˜19% reduction versus activity of the FL-PEG-Prom) withoutsignificantly altering activity of the PEG-Prom in E11 cells. Incontrast, removal of the PEA3 site at −104 nt with retention of the TATAbox at position −24 and the AP1 site at +8 bp resulted in a reduction inbasal promoter activity in both E11 and E11-NMT cells. The activity ofthis mutant PEG-Prom was 15- and 4-fold lower, respectively, than theactivity of the FL-PEG-Prom in E11-NMT and E11 cells (FIG. 5). Ineffect, this promoter deletion eliminated the enhanced expression of thePEG-Prom in E11-NMT versus E11 cells, indicating that the PEA3 site at−104 is a primary determinant of the enhanced activity of PEG-3 inprogressed H5ts125transformed RE cells. Internal deletions at position−1167 to −536 and −1267 to −536 resulted in similar levels of luciferaseactivity in E11-NMT and E11 cells as observed with the deletion mutantcontaining a deletion at position −270. Internal deletions engineeredbetween −1167 to −142 and −1590 to −142 resulted in a further decreasein promoter activity in both E11 and E11-NMT cells, with the mostprofound effect apparent in E11-NMT cells (˜41% reduction in activity incomparison with the FL-PEG-Prom). In contrast, deletion of the promoterregions from −142, −536 or −1287 with retention of the remainder of thePEG-Prom completely abolished PEG promoter activity (FIG. 5). Theseresults implicate the PEA3 transcription site (at position −104), theAP1 transcription site (at position +8) and the TATA box (at position−24) as primary determinants of basal PEG-Prom activity in E11 andE11-NMT cells.

[0163] To examine further the role of the PEA3 site at position −104,the TATA box at position −24 and the AP1 site at position +8 in theregulation of PEG-3 promoter activity in E11 and E11-NMT cells anadditional series of mutant PEG-3 promoter luciferase constructs weregenerated (FIG. 6). Mutation in the AP1 site, with retention of the wtPEA3 and TATA sites, resulted in equivalent promoter activity in E11 andE11-NMT cells. This observation emphasizes the importance of the AP1site at position +8 in the PEG promoter in regulating elevated PEG-3transcriptional activity in E11-NMT versus E11 cells. An involvement ofthe PEA3 site at position −104 in defining PEG promoter activity wasalso demonstrated by analysis of a construct containing a mutated PEA3site at −104 with wild-type TATA (at position −24) and AP1 (at position+8) sites (FIG. 6). In this mutant, the level of activity of thepromoter was at a basal level and the activity was similar in E11 andE11-NMT cells. A similar basal promoter activity was also observed withtwo additional mutants, one containing mutant AP1 and PEA3 sites and awild-type TATA box and a mutant lacking the PEA3 site at position −104with wild-type TATA and AP1 sites. In contrast, a mutant lacking thePEA3 site at position −104 with a mutated TATA site and a wild-type AP1site at position +8 displayed no promoter activity. These resultsconfirm that both the AP1 site located at +8 and the PEA3 site atposition −104 are involved in the differential expression of thePEG-Prom in E11-NMT versus E11 cells. AP1 and PEA3 are majordeterminants of the differential expression of the PEG-Prom in E11-NMTversus E11 cells and basal PEG-Prom activity in E11 and E11-NMT cells.

[0164] Progressed E11-NMT Cells Display Enhanced Nuclear TranscriptionFactor Binding

[0165] Western blotting analysis was performed to determine the levelsof AP1/cJun and PEA3 protein in E11 and E11-NMT cells. With bothproteins the de novo level of expression was ˜1.5 to 2 fold higher inE11-NMT versus E11 cells (data not shown). EMSA were performed todetermine the DNA binding potential of the AP1 and PEA3 proteins and ifdifferent levels of binding complexes are present in E11-NMT versus E11cells (FIGS. 7A and 7B, respectively). Using a wild-type AP1oligonucleotide, the level of binding to AP1 was higher in E11-NMTversus E11 (FIG. 7A). The specificity of this binding to AP1 wasdemonstrated by competition with a 10- and a 100-fold molar excess ofunlabeled competitor and the absence of a DNA-protein complex when usinga mutant AP1 oligonucleotide (FIG. 7A). Direct confirmation of bindingof nuclear extracts to AP1 was provided by supershift assays using cJun(AP1) antibody (FIG. 7A). In contrast, no supershifted DNA-proteincomplexes were observed when an anti-actin antibody was used in place ofthe cJun (AP1) antibody. Similar results were obtained when a PEA3oligonucleotide was used in gel retardation assays (FIG. 7B). Enhancedbinding to PEA3 was observed with extracts from E11-NMT versus E11cells. No binding was observed with a mutated PEA3 oligonucleotide,unlabelled PEA3 competitor effectively inhibited binding to PEA3 andantibodies specific for PEA3, but not anti-actin antibodies, resulted insupershifted DNA-protein complexes in the EMSA (FIG. 7B). Theseexperiments demonstrate that E11-NMT cells contain elevated levels ofAP1 and PEA3 with the capacity to bind to their respective sites in thepromoter of PEG-3.

[0166] Ectopic Expression of cJun (AP1) and PEA3 in E11 CellsIndependently and Cooperatively Enhance PEG-Prom Activity

[0167] The studies described above suggested that AP1 and PEA3 sites inthe PEG-Prom were responsible for the differential activity of thispromoter in E11-NMT versus E11 cells. To directly determine if theproteins encoded by these transcription factors can alter the expressionof the FL-PEG-Prom in E11 cells transient transfection andpromoter-luciferase assays were performed (FIG. 8). Transfection of E11cells with an expression vector producing cJun resulted in adose-dependent increase in FL-PEG-Prom activity in E11 cells. Themaximum effect obtained was small, equaling only an ˜1.5-fold increasein cells not expressing the cJun expression plasmid. This stimulatoryeffect was not evident in cells transfected with a control vector(pcDNA3.1) or a vector encoding a mutant cJun protein (TAM67). Forcedexpression of PEA3 in E11 cells also resulted in a dose-dependentincrease in FL-PEG-Prom activity, again reaching a maximum of ˜1.5-fold.No enhancement in promoter activity was observed in E11 cellstransfected with the control pRC/RSV vector. When E11 cells wereco-transfected with a combination of expression vectors producing cJunand PEA3, FL-PEG-Prom activity was comparable to that observed inE11-NMT cells. This effect was not apparent when the combination ofcontrol vectors were transfected into E11 cells (FIG. 8). These resultsprovide support for the hypothesis that the differential expression ofthe PEG-Prom in E11-NMT versus E11 cells is a consequence of elevatedexpression of cJun (AP1) and PEA3 transcription factors in theprogressed E11-NMT cells.

[0168] Discussion

[0169] Acquisition of enhanced expression of the transformed phenotype,i.e., transformation progression, represents a critical component in thecancer paradigm. A novel cDNA, PEG-3, that displays differentialexpression as a function of progression of the transformed phenotype,oncogenic transformation and DNA damage in rodent cells was identifiedby subtraction hybridization (Su et al., 1997). Recent studies documentthat PEG-3 is causally related to cancer progression, since ectopicexpression of this gene in transformed rodent or human tumor cellsresults in an aggressive tumor phenotype when cells are injectedsubcutaneously into athymic nude mice (Su et al., 1999). Theseobservations suggest that PEG-3 is an important contributor totransformation progression. To define the mechanism mediatingdifferential expression of PEG-3 in progressed (E11-NMT) versusunprogressed (E11) Ad5-transformed rat embryo cells the promoter regionof this gene was identified, isolated and examined. By using promoteranalyses, EMSA and transient transfection assays we presentlydemonstrate that a combination of the AP1 and PEA3 transcription factorsites in the PEG-Prom adjacent to the TATA, region contribute to basaland enhanced promoter activity in H5ts125-transformed RE cells.

[0170] Promoter deletion analysis indicates that a region of thePEG-Prom containing −270/+194 of the PEG-3 gene is essential for PEG-3transcriptional activity in E11 and E11-NMT cells (FIGS. 5 and 6).Moreover, this region of the PEG-Prom is also responsible for thedifferential promoter activity of the PEG-Prom in E11-NMT versus E11cells. Sequence analysis indicates that this part of the PEG-Promcontains AP1 (+8), TATA (−24) and PEA3 (−104) elements (FIG. 2). Amutation of the AP1 site at +8, while retaining a wild-type TATA andPEA3 sequence, reduces the activity of the PEG-Prom deletion construct(−270/+194) in E11-NMT to that of E11 cells (FIG. 6B). This findingsuggests that the AP1 site at +8 is a primary determinant of thedifferential expression of the PEG-Prom in E11-NMT versus E11 cells. Theimportance of the TATA and PEA3 sites in PEG-Prom activity is alsodocumented using additional mutants (FIG. 6B). A mutation in the PEA3site (−104) in the presence of wild type TATA (−24) and AP1 (+8) sitesreduces promoter activity in E11 and E11-NMT and effectively eliminatesthe enhanced activity of the PEG-Prom in E11-NMT cells. Similar levelsof reduced PEG-Prom activity are apparent in both E11 and E11-NMT cellswhen the AP1 (+8) site is mutated singly or in combination with amutated PEA3 (+8) site. In these contexts, altering the AP1 (+8) andPEA3 (104) sites, singly or in combination, effects both basal andenhanced PEG-Prom activity. Moreover, a mutation in the TATA region(−24), even in the presence of a wild-type AP1 (+8) site, results in anextinction of promoter activity. These results demonstrate that both AP1and PEA3 sites adjacent to an intact TATA region within the PEG-Promcontribute to both basal promoter activity in E11 and E11-NMT cells andelevated promoter activity in E11-NMT cells.

[0171] A functional interaction between the AP1 and PEA3 sites andbinding of nuclear proteins in the FL-PEG-Prom was confirmed by EMSAusing appropriate oligonucleotide probes and monoclonal antibodies (FIG.7). EMSA using nuclear extracts from E11 and E11-NMT cells resulted inslower-migrating DNA-protein complexes when incubated with AP1 or PEA3oligonucleotides (FIGS. 7A and 7B). The amount of these complexes werereduced or eliminated when a 10-or 100-fold molar excess, respectively,of unlabelled oligonucleotides were incorporated in the assay. NoDNA-protein complexes were observed when a mutated AP1 or PEA3oligonucleotide was used in the binding assay. The specificity of thenuclear protein binding was demonstrated using antibody specific forcJun (AP1) or PEA3 in the EMSA. In these experiments supershiftedslow-migrating DNA-protein complexes were apparent resulting fromantibody interactions with the DNA-protein complexes. The amount of AP1and PEA3 complexes present in E11-NMT cells exceed that found in E11cells (FIGS. 7A and 7B). Moreover, a small but significant increase(˜1.5 to 2-fold) in the levels of AP1/cJun and PEA3 protein was alsodetected by Western blotting in E11-NMT versus E11 cells (unpublisheddata). The functional significance of the elevated AP1 and PEA3 proteinsin E11-NMT versus E11 cells in regulating elevated PEG-3 promoteractivity in the progressed cells was documented by transienttransfection of cJun and PEA3 expression vectors (FIG. 8). Theseexperiments demonstrated that transient ectopic cJun (AP1) and PEA3expression can individually elevate PEG-Prom activity in E11 cells andthe combination of both transcription factors results in an additiveeffect culminating in a similar PEG-Prom activity as observed in E11-NMTcells (FIG. 8). Based on increased binding activity in EMSA, increasedlevels of protein in Western blots and cotransfection assays thereappears to be a strong correlation between PEG-3 expression and AP1/PEA3activity.

[0172] AP1 transcription factors are immediate early response genes thatregulate expression of a subset of target gene promoters containingdefined sequence motifs (TPA-response elements, TRE) (Angel and Karin,1991). The AP1 complex comprises a heterodimer of a member of the Fosfamily and a member of the Jun family or homodimers of members of theJun family (Angel and Karin, 1991, Karin et al., 1997). AP1 contributesto many important and diverse biological processes including cellproliferation, transformation, onocogenesis, differentiation andapoptosis (Angel and Karin, 1991; Karin et al., 1997; Olive et al.,1997; Kang et al., 1998b). The transcription factor PEA3 a member of theets gene family is also a major contributor to cell transformation andoncogenesis (Brown and McKnight, 1992). PEA3 proteins interact with an˜10 base pair DNA sequence in the promoters of target genes resulting inregulation of transcription (Macleod et al., 1992; Seth et al., 1992;Wasylyk et al., 1993). Putative candidate PEA3 target genes includeproteinases required for degradation of the extracellular matrix,including the serine urokinase-type plasminogen activator (Nerlov etal., 1992) and matrix metalloproteinases gelatinase B, interstitialcollagenase, stromelysin-3 and matrilysin (Matrisian and Bowden, 1990;Matrisian, 1994; Higashino et al., 1995), which represent importantfactors contributing to cancer metastasis (Liotta et al., 1991; Kohn andLiotta, 1995). Many of these extracellular matrix degrading genes alsocontain AP1 sites in their promoters (Angel and Karin, 1991; Karin etal., 1997). Cooperation between AP1 and PEA3 sites in regulating severalcellular promoters have been documented. These include, serum growthfactor response of the tissue inhibitor of metalloproteinases-1 (TIMP-1)gene (Edwards et al., 1992) and 12-0-tetradecanoylphorbol 13-acetate(TPA), fibroblast growth factor-2 (FGF-2) and macrophagecolony-stimulating factor induction of the urokinase-type plasminogenactivator gene (Neriov et al., 1992; Stacey et al., 1995; De Cesare etal., 1996; D'Orazio et al., 1997). Moreover, PEA3 and AP1 elements arealso present in the promoters of the stromelysin and collagenase genes(Gutman and Wasylyk, 1990; Sirum-Conolly and Brinckerhoff, 1991) andthese elements provide targets for transcriptional activation byspecific transforming oncogenes (Wasylyk et al., 1989, 1993). In thesecontexts, the increased AP1 and PEA3 activity in E11-NMT cells versusE11 can result in elevated PEG-Prom activity and thereby increased PEG-3protein which can directly contribute to cancer aggressiveness,resulting in enhanced tumor growth in vivo in nude mice, in theprogressed tumor cells. The increased activity of AP1 and PEA3 inE11-NMT cells will also likely activate additional down-stream genesthat can facilitate the cancer phenotype.

[0173] The mechanism by which PEG-3 facilitates expression of thetransformed phenotype is not currently known. Forced expression of therat PEG-3 gene in both rodent and human cancer cells results in anincrease in anchorage independent growth and an augmentation inoncogenic potential (Su et al., 1997, 1999). One putative target forPEG-3 is the angiogenesis-inducing molecule, vascular endothelial growthfactor (VEGF) (Su et al., 1999). Stable elevated expression of PEG-3results in increased VEGF RNA transcription, steady-state mRNA andsecreted protein in E11 cells. Moreover, a VEGF-luciferase reporterconstruct displays enhanced activity in cells expressing PEG-3. Afunctional role for PEG-3 in regulating VEGF expression is demonstratedfurther by inhibiting PEG-3 expression in E11-NMT cells using a stableantisense PEG-3 expression vector which results in a decrease in VEGFmRNA and secreted protein. The requirement for PEG-3 protein in inducingVEGF expression was demonstrated by simultaneous treatment of PEG-3transfected cells with the protein synthesis inhibitor cycloheximide (Suet al., 1999). In this experiment, the transtected PEG-3 gene wasexpressed as PEG-3 mRNA, whereas VEGF mRNA was only present in cells notexposed to cycloheximide. Although it is not presently known if PEG-3binds directly to the VEGF promoter or activation of VEGF transcriptionoccurs by means of additional molecules, these studies suggest anassociation between PEG-3 expression, induction of angiogenesis andfacilitation of expression of the cancer state.

[0174] Further studies are necessary to identify and characterize therepertoire of down-stream genes modulated as a consequence of PEG-3expression and to determine their roles in facilitating canceraggressiveness and angiogenesis. These investigations are important andoffer potential for defining the genetic elements which are criticaldeterminants of the cancer phenotype. With this information it will bepossible to distinguish potential targets and define appropriatereagents, such as antisense or small molecule antagonists, forinhibiting or preventing cancer development and progression.

[0175] Materials and Methods

[0176] Cell Cultures

[0177] E11 is a single cell clone of H5ts125-transformed Sprague-Dawleysecondary RE cells (Fisher et al., 1978). E11-NMT is a subclone of E11cells derived from a nude mouse tumor induced by the E11 cell line(Babiss et al., 1985). R12 is a Ha-ras oncogene transformed E11 clone(Duigou et al., 1989). F1 and F2 are suppressed somatic cell hybridswith a flat morphology that were formed between E11-NMT and CREF cells(Duigou et al., 1990). R1 and R2 are progressed somatic cell hybridswith a round morphology that were created by fusing E11-NMT and CREFcells (Duigou et al., 1990). CREF is a specific immortal non-transformedand non-tumorigenic clone of Fischer rat embryo fibroblast cells (Fisheret al., 1982). All cultures were grown in Dulbecco's modified Eagle'smedium (DMEM) supplemented with 5% FBS (DMEM-5) at 37° C. in ahumidified 5% CO₂ 95% air incubator.

[0178] Northern and Western Blotting Assays.

[0179] Total cellular RNA was isolated by the guanidinium/phenolextraction method and Northern blotting was performed as described (Suet al., 1994, 1997). Fifteen μg of RNA were denatured andelectrophoresed in 1.2% agarose gels with 3% formaldehyde, transferredto nylon membranes and hybridized sequentially with ³²P-labeled cDNAprobes as described previously (Su et al., 1994, 1997). Followinghybridization, the filters were washed and exposed for autoradiography.Western blotting analyses (Su et al., 1995) detected cJun (AP1), PEA3,PEG-3 and actin proteins. Five million cells were seeded into 100-mmplates and incubated for 24 h at 37° C. The medium (DMEM-5) was removed,the cells were washed 3× with cold PBS and then lysed in RIPC buffer(0.5 M NaCl, 0.5% NP40, 20 mM Tris-HCI, pH 8, 1 mM PMSF). The proteinlevels were determined using an ECL kit (Amersham) and the respectiveantibodies (Santa Cruz). Cell lysates were also analyzed using rabbitanti-PEG-3 polyclonal antibodies against C-terminal peptides.

[0180] Isolation and Analysis of the PEG-3 Promoter

[0181] Based on the 5′ sequence of the PEG-3 cDNA, two nested primerswith the sequences GATCTAGGGTGTTGTGAGAGGATCGGAG (SEQ ID NO:2) andTCGGTTTGCCAAAAGCGATCGTGGG (SEQ ID NO:3) were used with a Genome WalkerKit (Clontech) to obtain a genomic sequence containing the putativepromoter of PEG-3. Three DNA fragments of 2.0-, 1.6- and 1.0-kb,respectively, with identical and overlapping nucleotide sequences wereobtained using this approach. The 2.0-kb PEG-3 fragment (designatedFL-PEG-Prom) was cloned into the pGL3-basic Vector (Promega) forpromoter activity analysis. 5′-Deletion mutations in the FL-PEG-Promwere made with exonuclease III digestion using the Erase-A-Base System(Promega). 3′-Deletion mutations of the FL-PEG-Prom were made bydigestion with BstEll/Xhol, Sacll/Xhol and Ndel/Xhol, respectively.BstEll, Sacll and Ndel are 20 single-cut restriction endonucleasesrecognizing DNA sequences in the FL-PEG-Prom, Xhol restriction site islocated in the MCS of pGL3 vector near the 3′ end of the FL-PEG-Prom.The internal deletions were performed by digesting the FL-PEG-Prom withNdel/Sacll, Ndel/BstEll, Stul/BstEll and BstXl, respectively. Mutationsin the AP1-binding site, PEA3-binding site, and TATA box were made usinga sitespecific mutagenesis method with the Altered Sites 11 In VitroMutagenesis System (Promega). The PEG-Prom deletion mutants were clonedinto the pGL3-basic Luciferase Reporter Vector (Promega,). To evaluatethe activity of the various PEG-Prom-luciferase constructs, cells wereseeded at 2×10⁵/35-mm tissue culture plate and ˜24 h later transfectedwith 5 μg of the various PEG-Prom-luciferase constructs plus 1 μg ofSV40-β-gal Vector (Promega) mixed with 10 μl of Lipofectamine Reagent(Gibco) in 200 μl of serum-free media. After 20 min at RT, 800 μl ofserum-free media were added resulting in a final volume of 1 ml. Thetransfection mixture was removed after 14 hr and the cells were washed3× with serum-free media and incubated at 37° C. for an additional 48 hrin complete growth media. Cells were harvested and lysed to makeextracts (Gopalkrishnan et al., 1999) utilized in β-gal and Luciferasereporter assays. Luminometric determinations of Luciferase and Pgalactivity was performed using commercial kits (Promega and Tropix,respectively). For Luciferase assays, 10 μl of cell lysate were mixedwith 40 μl of Luciferase Assay substrate (Promega). For β-gal assays, 10μl of the cell lysate were mixed with 100 μl of diluted Galecton-Pluswith 150 μl of Accelerator (Tropix). Promoter analysis data werecollected a minimum of three times using triplicate samples for eachexperimental point and the data was standardized with the β-gal data.

[0182] Primer Extension of E11 and E11-NMT mRNA

[0183] A primer with the sequence 5′ GGCAAAGGGATGCGGAGTCGCGCGGGTCTCGCATG3′ (SEQ ID NO:4) complementary to the 5′ UTR sequence of the PEG-3 cDNAwas annealed to 4 μg of PolyA⁺ RNAs from E-11 or E11-NMT cells, whichwere used as template for primer extension with reverse transcriptase.In brief, 20 pmol of dephosphorylated oligo-DNA was end-labeled withγ-³²P ATP (Amersham) and T4 polynucleotide kinase. The labeledoligonucleotides (5×10⁵ cpm) were incubated with 4 μg of polyA+ RNA andthe precipitate was resuspended in DEPC-treated H₂O. The reversetranscription reaction contained 200 u/μl of Superscript ReverseTranscriptase II (Gibco), 50 mM of Tris-HCI (pH 8.3), 40 mM KCI, 6 mMMgC′21 1 mM DTT, 1 mM dNTP, and 0.1 mg/ml BSA. The mixture was incubatedat 42° C. for 1 hr followed by the addition of 1 ml of 0.5 M EDTA (pH 8)to stop the reaction. After DNase-free RNase treatment, the reactionmixture was loaded onto a 5% urea polyacrylamide sequencing gel inparallel with a DNA sequencing reaction using the same primer andtemplate.

[0184] Electrophoretic Mobility Shift Assays (EMSA)

[0185] Nuclear extracts were prepared from 2 to 5×10⁸ cells as describedby Dignam et al. (1983). The sequence of probes were as follows:wild-type AP1, 5′ CGCAGATTGACTCAGTTCGC3′ (SEQ ID NO:5)/5′GCGTCTAACTGAGTCAAGCG3′ (SEQ ID NO:6); mutant AP1, 5′CGCAGATAAACTACGTTCGC3′ (SEQ ID NO:7)/5′ GCGTCTATTTGATGCAAGCG3′ (SEQ IDNO:8); wild-type PEA3, 5′ GTGTTGTTTTCCTCTCTCCA3′ (SEQ ID NO:9)/5′CACAACAAAAGGAGAGGT3′ (SEQ ID NO:10); and mutant PEA3′, 5′GTGTTGTTCCCATCTCTCCA3′ (SEQ ID NO:11)/5′ CACAACA AGGGTAGAGAGGT3′ (SEQ IDNO:12). The double-stranded oligonucleotides were labeled with ³²P-ATP(Amersham) and T4 polynucleotide kinase. The labeled probes were thenincubated with nuclear extract at RT for 30 min. The reaction mixtureconsisted of ³²P-labeled deoxyoligonucleotides (>5000 cpm), 2 μg ofpoly(dl-dc) and 10 μg of nuclear protein extract with 10 mM HEPES (pH7.5), 50 mM KCI, 5 mM MgCI2, 0.5 mM EDTA, 1 mM DTT and 12.5% glycerol.After incubation for 30 min at RT, the reaction mixtures wereelectrophoresed on a 5% polyacrylamide gel with 0.5×TBE (160V for 3 h).The gel was dried and autoradiographed. Nuclear extracts were alsoincubated with a 10- or 100-fold molar excess of cold competitoroligonucleotide or cJun (AP1), PEA3 or actin antibody (1 or 5 μg)together with the ³²P-labeled probe.

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1 12 1 1970 DNA Rattus norvegicus promoter (1507)...(1970) PEG-3Promoter (corresponds to -270 to +194 of Figure 2) 1 acatgggcacgcgtggtcga cggcccgggc tggctgggca acacgggttc agcccaggtt 60 tcatagtaagttccagacac tcctggaaaa acaatacagg tccctgacaa aagaaaaaac 120 aaaacaaaggaaacagaaac atgcgttttt aaaaaagaag gaggagactc catgaaggca 180 ggccttgggtggggtcactg cttctctgta cacaggagga gaattgccaa gatcttccgg 240 acagtgtggactatactgta agaccctctc aatacagaca gactggacag gcatagtgac 300 acatgcctttaatgcctgca gtactcagga ggaggtggca ggtggaacgg ctgttctttg 360 aggttcaagaccagcgtgga ctacagagtg agttccagga caggcagggc tacacagaaa 420 aatcctgtctgaaaacaaaa caaaacccag acagacacac caaaaacagc caagggacca 480 gagagatgggtcagggccta atcacttgct actctttgca gaggacccaa atttagttcc 540 tataaccctccatgagaagc ttcacaattg tctctaactc aattccaccc gtgttccgac 600 ctcccatatgcaccagacat gttatactca cacatacgca caaacacaca cacacacaca 660 cacacacacacacacacaca cacacacaca cggaaaacat ataaaataaa gatttaaaaa 720 atctttttcttttggccggg gtgtgtggga gagcatctga gccatctcac cagcccaggg 780 tgcacgtctttttctttttt tcggagctgg ggaccgaacc cagagccttg tgcttgctag 840 gcaagtgctctaccactgag ctaaatcccc aaccccggag cacgtcttta atcccagaat 900 caggaggtagaggtaatgag atcccagtga gcccaaggtc agccgagtct acaaagtgag 960 ttccaggacagccagaacta atcttggaaa aacaaacaag ggctggtgag gtggttcagt 1020 agttaagaacactggctgct cttccagagg tcctgagttc attctcagta accacatggt 1080 ggggatctgatgcctgttct ggcatgcaga tatacatgca gatagtgcac tcctacattt 1140 aaaaaaaaaagacataaata atattttaaa acattgggcg ttttgtcttc taataaaact 1200 tcactgctatcttctaataa aaattcactg ctagccgcgg ggtgtggtgc ccccatacct 1260 ttaatcccaacaacttgaga ggcagaggca ggcggacctt tgagtttgaa gctagcctgg 1320 tctacagagtgagttcaaga tagccacgga tagtcagaaa gtcctgtttc gaacctctcc 1380 ccaaccaaatcactcctgta atcccagcac tctggaggca gtagcaggtt agtccctgct 1440 tctcagagagaggagagaga gagagagaga gaggagacac acacacacag agacagagag 1500 gagagagaaagagaaagaga atgggacagc atgtgactgc ctgatgaagt tggcgtgctt 1560 gctcaaaagttctgcgagat tgacggctct ctggatttga gccaaggaca cgcctgggaa 1620 gccacggtgacctcacaagg cccggaatct ccgcgagaat ttcagtgttg ttttcctctc 1680 tccacctttctcagggactt ccgaaactcc gcctctccgg tgacgtcagc atagcgctgc 1740 gtcagactataaactcccgg gtgatcgtgt tggcgcagat tgactcagtt cgcagcttgt 1800 ggaagattacatgcgagacc ccgcgcgact ccgcatccct ttgccgggac agcctttgcg 1860 acagcccgtgagacatcacg tccccgagcc ccacgcctga gggcgacatg aacgcgctgg 1920 ccttgagagcaatccggacc cacgatcgct tttggcaaac cgaaccggac 1970 2 28 DNA ArtificialSequence synthetic oligonucleotide 2 gatctagggt gttgtgagag gatcggag 28 325 DNA Artificial Sequence synthetic oligonucleotide 3 tcggtttgccaaaagcgatc gtggg 25 4 35 DNA Artificial Sequence syntheticoligonucleotide 4 ggcaaaggga tgcggagtcg cgcgggtctc gcatg 35 5 20 DNAArtificial Sequence synthetic oligonucleotide 5 cgcagattga ctcagttcgc 206 18 DNA Artificial Sequence synthetic oligonucleotide 6 gtctaactgagtcaagcg 18 7 19 DNA Artificial Sequence synthetic oligonucleotide 7cgcagataaa ctagttcgc 19 8 20 DNA Artificial Sequence syntheticoligonucleotide 8 gcgtctattt gatgcaagcg 20 9 20 DNA Artificial Sequencesynthetic oligonucleotide 9 gtgttgtttt cctctctcca 20 10 20 DNAArtificial Sequence synthetic oligonucleotide 10 cacaacaaaa ggagagaggt20 11 20 DNA Artificial Sequence synthetic oligonucleotide 11 gtgttgttcccatctctcca 20 12 20 DNA Artificial Sequence synthetic oligonucleotide 12cacaacaagg gtagagaggt 20

1-14 (Cancelled)
 15. A method for identifying an agent which modulatesPEG-3 promoter activity in a cell which comprises: (a) contacting thecell with the agent wherein the cell comprises a nucleic acid comprisinga PEG-3 promoter operatively linked to a reporter gene; (b) measuringthe level of reporter gene expression in the cell; and (c) comparing theexpression level measured in step (b) with the reporter gene expressionlevel measured in an identical cell in the absence of the agent, whereina lower expression level measured in the presence of the agent isindicative of an agent that inhibits PEG-3 promoter activity and whereina higher expression level measured in the presence of the agent isindicative of an agent that enhances PEG-3 promoter activity, therebyidentifying an agent which modulates PEG-3 promoter activity in thecell.
 16. The method of claim 15, wherein the cell is a melanoma cell, aneuroblastoma cell, a cervical cancer cell, a breast cancer cell, a lungcancer cell, a prostate cancer cell, a colon cancer cell or aglioblastoma multiforme cell.
 17. The method of claim 15, wherein theagent comprises a molecule having a molecular weight of about 7kilodaltons or less.
 18. The method of claim 15, wherein the agent is anantisense nucleic acid comprising a nucleotide sequence complementary toat least a portion of the sequence shown in SEQ ID NO:1 and is at least15 nucleotides in length.
 19. The method of claim 15, wherein the agentis a DNA molecule, a carbohydrate, aglycoprotein, a transcription factorprotein or a double-stranded RNA molecule.
 20. The method of claim 15,wherein the agent is a synthetic nucleotide sequence, a peptidomimetic,or an organic molecule having a molecular weight from
 0. 1 kilodaltonsto 10 kilodaltons.
 21. The method of claim 15, wherein the reporter geneencodes beta-galactosidase, luciferase, chloramphenicol transferase oralkaline phosphatase.
 22. The method of claim 15, wherein expression ofPEG-3 promoter activity measured is equal to or greater than a 2.5 to3.5 fold increase or decrease.
 23. The method of claim 15, wherein thePEG-3 promoter is a PEG-3 promoter having promoter activity comprisingthe nucleotide sequence beginning with the guanosine (G) at position1507 and ending with the cytosine (C) at position 1970 of SEQ ID NO:1.24. A method for treating cancer in a subject which comprisesadministering a nucleic acid comprising a PEG 3 promoter operativelylinked to a gene-of-interest wherein the gene of interest is selectivelyexpressed in cancerous cells in the subject and such expressionregulates expression of PEG-3 resulting in growth suppression or deathof the cancerous cells, thereby treating cancer in the subject.
 25. Themethod of claim 24, wherein the nucleic acid consists essentially of (i)a PEA3 protein binding sequence consisting of the nucleotide sequencebeginning with the thymidine (T) at position 1672 and ending with thethymidine (T) at position 1677 of SEQ ID NO:1, (ii) a TATA sequenceconsisting of the nucleotide sequence beginning with the thymidine (T)at position 1748 and ending with the adenosine (A) at position 1753 ofSEQ ID NO:1, and (iii) an AP1 protein binding sequence consisting of thenucleotide sequence beginning with the thymidine (T) at position 1781and ending with the adenosine (A) at position 1787 of the nucleotidesequence shown in SEQ ID NO:1.
 26. The method of claim 24, wherein thenucleic acid has a sequence complementary to at least a portion of SEQID NO: 1 of at least 25 nucleotides in length.
 27. The method of claim24, wherein the cancer is melanoma, neuroblastoma, astrocytoma,glioblastoma multiforme, cervical cancer, breast cancer, colon cancer,prostate cancer, osteoscarcoma or chrondosarcoma.
 28. The method ofclaim 24, wherein the administering is carried out via injection, oraladministration, topical administration, adenovirus infection,liposome-mediated transfer, topical application to the cells of thesubject, or microinjection.
 29. The method of claim 24, wherein thesubject is a mammal.
 30. The method of claim 29, wherein the mammal is ahuman.
 31. The method of claim 24, wherein the gene of interest is agene whose expression causes apoptosis of a cell.
 32. The method ofclaim 24, wherein the gene comprises an Mda-7 gene or a p53 gene. 33.The method of claim 24, wherein the gene of interest is a tumorsuppressor gene.
 34. The method of claim 33, wherein the suppressor geneis mda-7.
 35. The method of claim 24, wherein the gene of interest is acytotoxic gene.
 36. The method of claim 35, wherein expression of thecytotoxic gene causes cell death.
 37. The method of claim 36, whereinthe cytotoxic gene is selected from the group consisting of HSV-TK, p21,p27, and p10.